Antiplatelet Therapy Combined with Anastrozole Induces Features of Partial EMT in Breast Cancer Cells and Fails to Mitigate Breast-Cancer Induced Hypercoagulation.

Thromboembolic complications are a leading cause of morbidity and mortality in cancer patients. Cancer patients often present with an increased risk for thrombosis including hypercoagulation, so the application of antiplatelet strategies to oncology warrants further investigation. This study investigated the effects of anastrozole and antiplatelet therapy (aspirin/clopidogrel cocktail or atopaxar) treatment on the tumour responses of luminal phenotype breast cancer cells and induced hypercoagulation. Ethical clearance was obtained (M150263). Blood was co-cultured with breast cancer cell lines (MCF7 and T47D) pre-treated with anastrozole and/or antiplatelet drugs for 24 h. Hypercoagulation was indicated by thrombin production and platelet activation (morphological and molecular). Gene expression associated with the epithelial-to-mesenchymal transition (EMT) was assessed in breast cancer cells, and secreted cytokines associated with tumour progression were evaluated. Data were analysed with the PAST3 software. Our findings showed that antiplatelet therapies (aspirin/clopidogrel cocktail and atopaxar) combined with anastrozole failed to prevent hypercoagulation and induced evidence of a partial EMT. Differences in tumour responses that modulate tumour aggression were noted between breast cancer cell lines, and this may be an important consideration in the clinical management of subphenotypes of luminal phenotype breast cancer. Further investigation is needed before this treatment modality (combined hormone and antiplatelet therapy) can be considered for managing tumour associated-thromboembolic disorder.

Acetophenone Azine.

Acetophenone azine (CAS 729-43-1) was recently discovered as a potent allergen in shin pads and footwear containing the foam elastomer ethyl vinyl acetate. The compound is probably not intentionally added to ethyl vinyl acetate but is likely the result of reactions between other additives that take place during the manufacturing process. A patch testing concentration of 0.1% in acetone or petrolatum is recommended. Acetophenone azine should be part of patch testing shoe series, as well as plastics and glues series. The compound is not currently available from patch testing materials suppliers, a situation that hopefully will be corrected.

LC-MS analyses of N-acetyl-p-benzoquinone imine-adducts of glutathione, cysteine, N-acetylcysteine, and albumin in a plasma sample: A case study from a patient with a rare acetaminophen-induced acute swelling rash.

Acetaminophen (Paracetamol, APAP) has been widely used for many decades as an analgesic and antipyretic agent but APAP overdose often causes acute adverse reactions, particularly liver damage. The metabolically oxidized form of APAP, N-acetyl-p-benzoquinone imine (NAPQI), is chemically reactive and binds covalently to proteins. Therefore, NAPQI is believed to be the key metabolite that causes hepatotoxicity, especially under conditions of glutathione depletion. Other APAP-induced adverse reactions, such as skin damage, are rare and remain poorly studied. Here, we report a case study of a male patient who presented with an acute swelling skin rash (without hepatotoxicity) caused by therapeutic doses of APAP. Plasma samples were collected at 17 hr after dosing (during the manifestation of symptoms) and at one month (after recovery) and were subjected to LC-MS analysis of NAPQI-adducts. A significant concentration of NAPQI-cysteine adduct (33 pmol/mL) was found together with low concentrations of NAPQI-N-acetylcysteine adduct (2.0 pmol/mL) and NAPQI-glutathione adduct (0.13 pmol/mL). However, the NAPQI-albumin adduct was below the detection limit (below 0.001% modification on albumin) despite a previous report of high concentrations of NAPQI-albumin adduct following acute liver injury. Therefore, the observed APAP-induced skin damage may have had a different cause from APAP-induced liver injury.

Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos.

Strobilurins is the most widely used class of fungicides, but is reported highly toxic to some aquatic organisms. In this study, zebrafish embryos were exposed to a range concentrations of three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) for 96 h post-fertilization (hpf) to assess their aquatic toxicity. The 96-h LC50 values of pyraclostrobin, trifloxystrobin and picoxystrobin to embryos were 61, 55, 86 µg/L, respectively. A series of symptoms were observed in developmental embryos during acute exposure, including decreased heartbeat, hatching inhibition, growth regression, and morphological deformities. Moreover, the three fungicides induced oxidative stress in embryos through increasing reactive oxygen species (ROS) and malonaldehyde (MDA) contents, inhibiting superoxide dismutase (SOD) activity and glutathione (GSH) content as well as differently changing catalase (CAT) activity and mRNA levels of genes related to antioxidant system (Mn-sod, Cu/Zn-sod, Cat, Nrf2, Ucp2 and Bcl2). In addition, exposure to the three strobilurins resulted in significant upregulation of IFN and CC-chem as well as differently changed expressions of TNFa, IL-1b, C1C and IL-8, which related to the innate immune system, suggesting that these fungicides caused immunotoxicity during zebrafish embryo development. The different response of enzymes and genes in embryos exposed to the three fungicides might be the cause that leads to the difference of their toxicity. This work made a comparison of the toxicity of three strobilurins to zebrafish embryos on multi-levels and would provide a better understanding of the toxic effects of strobilurins on aquatic organisms.

Simulation of interindividual differences in inactivation of reactive para-benzoquinone imine metabolites of diclofenac by glutathione S-transferases in human liver cytosol.

Diclofenac is a widely prescribed NSAID that causes severe idiosyncratic drug induced liver injury (IDILI) in a small part of the patient population. Formation of protein-reactive metabolites is considered to play a role in the development of diclofenac-induced IDILI. Therefore, a high hepatic activity of enzymes involved in bioactivation of diclofenac is expected to increase the risk for liver injury. However, the extent of covalent protein binding may also be determined by activity of protective enzymes, such as glutathione S-transferases (GSTs). This is supported by an association study in which a correlation was found between NSAID-induced IDILI and the combined null genotypes of GSTM1 and GSTT1. In the present study, the activity of 10 different recombinant human GSTs in inactivation of protein-reactive quinoneimine (QI) metabolites of diclofenac was tested. Both at low and high GSH concentrations, high activities of GSTA1-1, A2-2, A3-3, M1-1, M3-3 and P1-1 in the inactivation of these QIs were found. By using the expression levels of GSTs in livers of 22 donors, a 6-fold variation in GST-dependent inactivation of reactive diclofenac metabolites was predicted. Moreover, it was shown in vitro that GSTs can strongly increase the efficiency of GSH to protect against the alkylation of the model thiol N-acetylcysteine by reactive diclofenac metabolites. The results of this study demonstrate that variability of GST expression may significantly contribute to the inter-individual differences in susceptibility to diclofenac-induced liver injury. In addition, expression levels of GSTs in in vitro models for hepatotoxicity may be important factors determining sensitivity to diclofenac cytotoxicity.

Acetophenone azine: a new allergen responsible for severe contact dermatitis from shin pads.

BACKGROUND: Contact dermatitis resulting from the use of shin pads is usually caused by rubber components, dyes, benzoyl peroxide, or formaldehyde resins. OBJECTIVES: To investigate and identify a new allergen in shin pads that was responsible for severe contact dermatitis in a young football player. METHODS: High-performance liquid chromatography (HPLC) of samples of shin pads was performed. The boy was patch tested with pieces of shin pads and with acetophenone azine, a chemical substance identified by HPLC in the foam of the shin pads. RESULTS: HPLC identified acetophenone azine at concentrations of approximately 20 µg/g of shin pad samples. Patch tests gave strongly positive reactions to pieces of shin pads and to acetophenone azine down to 0.001% in acetone, whereas acetophenone and hydrazine sulfate were both negative. Twenty controls were negative for acetophenone azine 0.01% in acetone. CONCLUSIONS: Acetophenone azine is a new, strong allergen of shin pads, and more generally of other sport equipment based on ethylene vinyl acetate. It may be used as a biocide, but this has to be confirmed. Further investigations are needed to understand factors such as exposure, cross-reaction patterns, metabolism, and the optimal patch test preparation.

Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury.

UNLABELLED: Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours. CONCLUSION: MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored.

Bioefficacy, residue dynamics and safety assessment of the combination fungicide trifloxystrobin 25% + tebuconazole 50%-75 WG in managing early blight of tomato (Lycopersicon esculentum Mill.).

This paper reports the in vitro and in vivo bioefficacy of a combination fungicide trifloxystrobin (25%) + tebuconazole (50%) against early blight disease of tomato (Lycopersicon esculentum Mill.) caused by Alternaria solani and their corresponding pre-harvest intervals (PHI) with reference to the maximum residue limits (European Union). Bioefficacy of the test fungicide combination revealed that in vitro conditions manifested the best control (75.1%) at 350 mg kg(-1) against 76.2% control under field conditions. A sample preparation method based on ethyl acetate extraction and estimation by LC-MS multiple reaction monitoring (MRM) was validated in tomato fruits at 0.01 mg/kg and dissipation studies were conducted in field at single and double doses. The residues of both the compounds on all the sampling days were below the European Union maximum residue limits (EU-MRLs) and the maximum permissible intakes (MPIs) were calculated on the basis of prescribed acceptable daily intake (ADI). The combined bioefficacy and residue dynamics information will support label-claim of this fungicide combination for the management of early blight in tomato.

Chromone linked nitrone derivative induces the expression of iNOS2 and Th1 cytokines but reduces the Th2 response in experimental visceral leishmaniasis.

In our previous work we have shown that the novel synthetic chromone derivative could effectively inhibit the Leishmania donovani replication in vitro and in vivo with less cytotoxicity on murine splenocytes. The aim of the present study is to explore the possible mechanism of anti-leishmanial effect of C-(6-methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone (designated as NP1) in vitro and in vivo in experimental visceral leishmaniasis caused by L. donovani. The cytotoxic effect of this derivative was studied in murine peritoneal macrophages by MTT method. NP1 at a dose of 17.06 µM showed 50% inhibition on L. donovani promastigotes but found less cytotoxic to the RAW 264.7 cells. Even the higher concentration of IC50 (up to four fold) did not exert much cytotoxic effect on RAW 264.7. Interestingly, NP1 at lower concentration (8.53 µM) could inhibit 50% of intracellular amastigotes in murine peritoneal macrophages. L. donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. NP1 was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression at mRNA level and by up-regulating proinflammatory cytokines such as IL-12 and IFN-γ and limiting the expression of IL-10 in vivo. The NO dependent killing was further confirmed in iNOS(-/-) mice compared to wild type. In agreement with the fact, induced synthesis of IL-12 and IFN-γ and associated down-regulation of IL-10 by the treatment of NP1 clearly indicated the possibility of novel strategy of drug development against Leishmania infection.

PAR-1 antagonists: current state of evidence.

Vorapaxar (SCH 530348) and atopaxar (E5555) are oral protease-activated receptor-1 (PAR-1) antagonists with high bioavailability. They inhibits thrombin induced platelet aggregation by competitively inhibiting PAR-1. We systematically evaluated the evidence for the efficacy and safety of all PAR-1 antagonists as well as for the individual drugs vorapaxar and atopaxar in different databases-PubMed, EMBASE, Scopus, and Cochrane register of Controlled Clinical Trials (CENTRAL).We selected randomized controlled trials of PAR-1 antagonists that reported on cardiovascular mortality as a clinical outcome. The random-effects Mantel-Haenszel model was used to evaluate the effect of PAR-1 antagonists on cardiovascular mortality. Seven trials were selected (N = 42,355) for analysis. PAR-1 antagonists as a class, as well as individually, were associated with a non-significant numerically lower risk of cardiovascular mortality than that seen with agents used in the control group; RR, 0.93; 95% CI, 0.83-1.04; P = 0.20). No heterogeneity was noted. However, PAR-1 antagonists also appeared to increase the risk of bleeding significantly. PAR-1 antagonists appear to be associated with some reduction in the risk of cardiovascular mortality; however the significantly higher bleeding risk noted with PAR-1 antagonists appear to mandate a very careful selection of patients that may benefit without a substantially increased risk of bleeds.

Risk of intracranial hemorrhage with protease-activated receptor-1 antagonists.

BACKGROUND AND PURPOSE: Recent clinical trial data suggest that protease-activated receptor-1 (PAR-1) antagonists may increase the risk of intracranial hemorrhage. Our objective was to investigate the qualitative and quantitative risks of intracranial hemorrhage in patients receiving PAR-1 antagonist therapy. METHODS: Pubmed, EMBASE, Cochrane Central Register of Controlled Trials, and Clinicaltrials.gov from 1966 to May 2012, were searched to identify relevant studies. We included randomized controlled trials that included a comparison of PAR-1 antagonist with placebo and in which the total number of patients and intracranial hemorrhage events were reported separately for active treatment and control groups. Summary incidence rates, relative risks, and 95% confidence intervals (CIs) were calculated using random-effects models. Between-study heterogeneity was assessed using the I2 statistic. RESULTS: In 9 PAR-1 antagonist trials with 42000 patients with a history of thrombotic vascular disease or acute coronary syndrome, PAR-1 antagonist treatment was associated with increased risk of intracranial hemorrhage (0.59% vs 0.30%; relative risk, 1.98; 95% CI, 1.46-2.68; P<0.00001; number needed to harm, 345). There was no heterogeneity across trials (P=0.84; I2=0%), PAR-1 antagonist agent (P=0.52), treatment duration (P=0.38), or trial-qualifying event (P=0.59). Risk of death from any cause or a cardiovascular cause did not differ between active treatment and control groups. CONCLUSIONS: In a pooled analysis of data from 9 trials, PAR-1 antagonist therapy was associated with an increased risk for intracranial hemorrhage.

Atopaxar. A novel player in antiplatelet therapy?

Atopaxar, also known as E 5555 is a novel reversible protease-activated receptor-1 (PAR-1) thrombin receptor antagonist. To date, Atopaxar has been investigated in phase II trials with focus on safety and tolerability in patients with acute coronary syndromes or stable coronary artery disease on top of standard antiplatelet therapy. Atopaxar was generally well tolerated, however a rise in liver enzymes and prolongation of the QTcF interval were observed. The data suggest, that atopaxar administration may promote some minor bleeding complications, but does not seem to significantly increase the risk of major bleeding. Although not powered for efficacy, the currently available data suggest potential benefits in patients at high risk for recurrent ischemic events on top of standard antiplatelet therapy. In conclusion, more studies (e.g. phase III) are needed to evaluate efficacy and safety of atopaxar.

Safety and efficacy of protease-activated receptor-1 antagonists in patients with coronary artery disease: a meta-analysis of randomized clinical trials.

BACKGROUND: Thrombin receptor antagonists blocking protease-activated receptor-1 (PAR-1) on platelets represent a new class of oral antiplatelet agents for patients with atherothrombotic disease manifestations. OBJECTIVES: We investigated the safety and efficacy of PAR-1 antagonists in patients with coronary artery disease (CAD). PATIENTS/METHODS: Randomized, placebo-controlled trials of the PAR-1 antagonists atopaxar or vorapaxar in CAD patients were identified. The primary safety endpoint was the composite of Thrombolysis In Myocardial Infarction (TIMI) clinically significant bleeding. The primary efficacy endpoint was the composite of death, myocardial infarction (MI) or stroke. RESULTS: A total of 41 647 patients from eight trials were included. PAR-1 antagonists were associated with higher risks of TIMI clinically significant (odds ratio [OR] 1.48, 95% confidence interval [CI] 1.39-1.57, P < 0.001), major (OR 1.46, 95% CI 1.28-1.67, P < 0.001) and minor (OR 1.67, 95% CI 1.40-2.00, P < 0.001) bleeding than placebo in the fixed-effects model. PAR-1 antagonists reduced the composite of death, MI or stroke as compared with placebo (OR 0.87, 95% CI 0.81-0.92, P < 0.001), driven by a lower risk of MI (OR 0.85, 95% CI 0.78-0.92, P < 0.001). Conversely, PAR-1 antagonists and placebo did not differ in terms of risk of death (OR 0.99, 95% CI 0.90-1.09, P = 0.81) or stroke (OR 0.96, 95% CI 0.84-1.10, P = 0.59). CONCLUSIONS: PAR-1 antagonists decrease ischemic events in patients with CAD as compared with placebo, mainly driven by a reduction in MI, at the cost of an increased risk of clinically significant bleeding.

Emerging antiplatelet therapy for coronary artery disease and acute coronary syndrome.

Antiplatelet therapy is used widely with proven benefit for the prevention of further ischemic cardiac complications in patients with known coronary artery disease (CAD) and a history of acute coronary syndrome (ACS). The limitations of conventional antiplatelet therapy with aspirin, clopidogrel, or prasugrel, as well as the fact that rates of recurrent ischemic events still remain high with use of these agents, underscore the need to investigate alternate agents that may further reduce event rates while limiting bleeding risk. The selection of antiplatelet therapy is further influenced by the following: ticagrelor was approved in July 2011 by the United States Food and Drug Administration (FDA), and clopidogrel is slated to become available as a generic productin 2012. We provide an overview of emerging agents for the treatment of CAD and ACS, including the reversible P2Y(12) antagonists ticagrelor, cangrelor, and elinogrel, and a new class of oral protease-activated receptor-1 (PAR-1) inhibitors, vorapaxar and atopaxar.The recently approved P2Y(12) antagonists prasugrel and ticagrelor demonstrate enhanced ability to prevent adverse cardiac outcomes. However, this comes at a cost of a potential increased risk of bleeding. New adverse effects have also emerged, including dyspnea for all of the reversible P2Y(12) antagonists (ticagrelor, cangrelor, and elinogrel) and ventricular pauses for ticagrelor. In addition, the newer P2Y(12) antagonists have a faster onset and offset. Two of these agents, cangrelor and elinogrel, are available as intravenous formulations, which may provide additional benefits in patients who undergo coronary artery bypass graft (CABG) surgery. Trials with the PAR-1 inhibitors have also shown trends toward reductions in cardiac events, but not without the possibility of increased bleeding. More than ever, as the arsenal of antiplatelet therapy expands, health care providers need to understand the pharmacologic and pharmacodynamic differences between conventional and emerging antiplatelet therapies for patients with ACS and CAD. Health care providers must also carefully assess patient-specific factors such as risk of thrombosis, concomitant disease states, age, drug adherence, and aspirin dose, and plan for those patients who will be undergoing CABG when selecting antiplatelet therapy in order to optimally balance bleeding and thrombosis risk.

Induction of apoptosis by survivin silencing through siRNA delivery in a human breast cancer cell line.

Post-transcriptional silencing of antiapoptotic genes is a promising strategy for cancer therapy, but delivering short interfering RNA (siRNA) molecules against such targets is challenging due to inability of anionic siRNA to cross cellular membranes. Lipid substitution on small molecular weight, nontoxic polyethylenimine (PEI) has been investigated as a promising approach for effective siRNA delivery. In this study, we report on the ability of low molecular weight, lipid-substituted PEI to deliver siRNA against the antiapoptotic protein survivin. Toxicity of a library of lipid-substituted PEIs, as well as their siRNA delivery and survivin silencing efficiency, was evaluated in MDA-MB-231 human breast cancer cells. A significant increase in cellular delivery of siRNA was observed as a result of lipid substitution. Most significant downregulation of survivin was established by caprylic acid-substituted polymers, which resulted in significant levels of apoptosis induction and resultant loss of cell viability. Survivin downregulation prior to anticancer drug treatment decreased the IC(50) of several drugs by 50- to 120-fold. Our experiments indicated an effective downregulation of survivin, a cell protective protein upregulated in tumor cells, by delivering siRNA with hydrophobically modified PEI. This study introduces a promising delivery system for safe and effective siRNA delivery that will be suitable for further investigation in preclinical animal models.

Randomized trial of atopaxar in the treatment of patients with coronary artery disease: the lessons from antagonizing the cellular effect of Thrombin­Coronary Artery Disease Trial.

BACKGROUND: Thrombin is a key mediator of platelet activation. Atopaxar is a reversible protease-activated receptor-1 antagonist that interferes with thrombin-mediated platelet effects. The phase II Lessons From Antagonizing the Cellular Effect of Thrombin-Coronary Artery Disease (LANCELOT-CAD) trial examined the safety and tolerability of prolonged therapy with atopaxar in subjects with CAD. METHODS AND RESULTS: Subjects with a qualifying history were randomized in a double-blind fashion to 3 dosing regimens of atopaxar (50, 100, or 200 mg daily) or matching placebo for 24 weeks and followed up for an additional 4 weeks. The key safety end points were bleeding according to the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) and Thrombolysis in Myocardial Infarction (TIMI) classifications. Secondary objectives included platelet aggregation and major adverse cardiac events. Seven hundred and twenty subjects were randomized. Overall bleeding rates tended to be higher with atopaxar compared with placebo by CURE criteria (placebo, 0.6%; atopaxar, 3.9%; relative risk, 6.82, P=0.03; 50 mg, 3.9%; 100 mg, 1.7%; 200 mg, 5.9%; P for trend=0.01) and TIMI criteria (placebo, 6.8%; atopaxar, 10.3%; relative risk, 1.52, P=0.17; 50 mg, 9.9%; 100 mg, 8.1%; 200 mg, 12.9%; P for trend=0.07). There was no difference in major bleeding. Major adverse cardiac events were numerically lower in the atopaxar subjects. All atopaxar regimens achieved high levels of platelet inhibition. A transient elevation in liver transaminases and dose-dependent QTc prolongation without apparent complications were observed in higher-dose atopaxar treatment groups. CONCLUSIONS: In this dose-ranging study of patients with CAD, treatment with atopaxar resulted in platelet inhibition, more minor bleeding, and numerically but not statistically fewer ischemic events. Larger-scale trials are needed to determine whether these patterns translate into clinically meaningful effects. CLINICAL TRIAL REGISTRATION: URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00312052.