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Since platelet aggregation is implicated in many pathological situations such as myocardial infarction, stroke, unstable angina and coronary artery disease, potent and safe antiplatelet agents would be useful for treating and preventing these diseases. Exposure of glycoprotein (GP)IIb/IIIa is the final common pathway leading to platelet activation and aggregate formation. Thus, the development of agents acting on this complex is an active area of research. Indeed, three GPIIb/IIIa antagonists are currently marketed but can only be intravenously administrated. Therefore, efforts are made to discover orally-active agents of this class, in order to use them for treatment of chronic disease or prevention of cardiovascular events. Although some clinical evaluation of orally-active compounds gave disappointing and discouraging results, research is still conducted to find compounds characterised by a better binding profile. This review describes 16 patents on new GPIIb/IIIa antagonists and more than 40 molecules characterised by strong activity against GPIIb/IIIa.

Maximal left ventricular (LV) hydraulic power output (PWRmax), corrected for preload as PWRmax/(Ved)β (where Ved is the end-diastolic volume and β is a constant coefficient), is an index of LV contractility. Whereas preload-adjusted maximal power (PAMP) is usually calculated with β = 2, there is uncertainty about the optimal value of β (β = 1 for the normal LV and 2 for the dilated LV). The aim of this work is to study the determining factors of β. The data set consisted of 245 recordings (steady state and vena cava occlusion) in 10 animals in an ischemic heart pig model. The occlusion data yielded the slope ( E es; 2.01 ± 0.77 mmHg/ml, range 0.71–4.16 mmHg/ml) and intercept (V0; −11.9 ± 22.6 ml; range −76 to 39 ml) of the end-systolic pressure-volume relation, and the optimal β-factor (assessed by fitting an exponential curve through the Ved-PWRmax relation) was 1.94 ± 0.88 (range 0.29–4.73). The relation of β with Ved was weak [β = 0.60 + 0.02(Ved); r 2 = 0.20]. In contrast, we found an excellent exponential relation between V0 and β [β = 2.16[Formula: see text] r 2 = 0.70]. PAMP, calculated from the steady-state data, was 0.64 ± 0.40 mW/ml2 (range 0.14–2.83 mW/ml2) with a poor correlation with E es ( r = 0.30, P< 0.001). An alternative formulation of PAMP as PWRmax/(Ved − V0)2, incorporating V0, yielded 0.47 ± 0.26 mW/ml2 (range 0.09–1.42 mW/ml2) and was highly correlated with E es ( r = 0.89, P< 0.001). In conclusion, correct preload adjustment of maximal LV power requires incorporation of V0 and thus of data measured under altered loading conditions.

ADP plays a crucial role in haemostasis and thrombosis and its receptors are potential target for antithrombotic drugs. The knowledge of the ADP-receptors has been amplified by recent discoveries. This review highlights the ADP-receptors models and their antagonists described in the recent literature, mainly the thienopyridine derivatives.

We studied the effects on pulmonary hemodynamics of U-46619, a thromboxane A2 (TXA2) agonist, before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Six anesthetized pigs (Ago group) received 6 consecutive injections of U-46619 at 30-min interval and were compared with six anesthetized pigs (Anta group) which received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. Consecutive changes in pulmonary hemodynamics, including characteristic resistance, vascular compliance, and peripheral vascular resistance, were continuously assessed during the experimental protocol using a four-element Windkessel model. At 2 mg/kg, BM-573 completely blocked pulmonary hypertensive effects of U-46619 but pulmonary vascular compliance still decreased. This residual effect can probably be explained by a persistent increase in the tonus of the pulmonary vascular wall smooth muscles sufficient to decrease vascular compliance but not vessel lumen diameter. Such molecule could be a promising therapeutic approach in TXA2 mediated pulmonary hypertension as it is the case in pulmonary embolism, hyperacute lung rejection and endotoxinic shock.

Arachidonic acid (AA) can be metabolised by various enzymes, most notably the cyclooxygenases and the lipoxygenases. The secondary messenger pathway leads to the formation of mediators that are implicated in pathologies such as asthma, inflammation and cancer. Leukotrienes (LTs) are produced through the lipoxygenase pathway. The observation that modulation of leukotriene production by inhibition of 5-lipoxygenase (5-LOX) leads to therapeutic benefit has encouraged research of potent 5-LOX inhibitors. As a consequence, many patents concerning 5-LOX inhibition and the uses of 5LOX inhibitors as therapeutic agents are claimed each year. This article reviews these patents for the period 1999 – 2002.

As scientists involved in the research and official registration of vaccines in Belgium and at the EU, we would like to reply to the paper by Lucija Tomljenovic and Christopher A. Shaw, ‘Human papi...

Prostanoids (prostaglandins and the thromboxanes) are cyclooxygenase products derived from C-20 unsaturated fatty acids. Since arachidonic acid is the most abundant among these precursor fatty acids in mammals, including humans, the series 2 prostanoids are predominantly formed in the body. Thus, cyclooxygenases metabolise arachidonate to five primary prostanoids: PGE2, PGF2 α, PGI2, TXA2 and PGD2. These lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP and DP, respectively. As a review focused on latest discoveries and developments of novel TXA2 modulators patented from January 1997 to December 2000 has been recently published, this paper specifically focuses on newly developed IP, DP, FP and EP modulators patented for three years, from January 1998 to December 2001. Indeed, because prostaglandins are involved in a large series of pathophysiological processes, a classification of these modulators has been proposed, bas...

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances that are involved in several physiological processes but also in pathological conditions, such as inflammation. It is actually well known that this enzyme exists under two forms: COX-1 and COX-2. Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations that COX-1 was involved in several homeostatic processes, while COX-2 expression was associated with inflammation and other pathologies, such as cancer proliferation, have led to the development of COX-2 selective inhibitors to improve the therapeutic potency and to reduce the classical side effects associated with the use of conventional NSAIDs. A large number of patents concerning COX inhibition are claimed each year and this article reviews patents claimed during the period January 1998 to December 2001.

A rapid and accurate diagnosis in patients with suspected heparin-induced thrombocytopenia (HIT) is essential for patient management but remains challenging. Current HIT diagnosis ideally relies on a combination of clinical information, immunoassay and functional assay results. Platelet activation assays or functional assays detect HIT antibodies that are more clinically significant. Several functional assays have been developed and evaluated in the literature. They differ in the activation endpoint studied; the technique or technology used; the platelet donor selection; the platelet suspension (washed platelets, platelet rich plasma or whole blood); the patient sample (serum or plasma); and the heparin used (type and concentrations). Inconsistencies in controls performed and associated results interpretation are common. Thresholds and performances are determined differently among papers. Functional assays suffer from interlaboratory variability. This lack of standardization limits the evaluation and the accessibility of functional assays in laboratories. In the present article, we review all the current activation endpoints, techniques and methodologies of functional assays developed for HIT diagnosis.

Silver nanoparticles (Ag NPs) are increasingly used in biomedical applications because of their large antimicrobial spectrum. Data in the literature on the ability of Ag NPs to perform their desired function without eliciting undesirable effects on blood elements are very limited and contradictory. We studied the impact of Ag NPs on erythrocyte integrity, platelet function and blood coagulation. Erythrocyte integrity was assessed by spectrophotometric measurement of haemoglobin release. Platelet adhesion and aggregation was determined by light transmission aggregometry and scanning electron microscopy. The calibrated thrombin generation test was used to study the impact on coagulation cascade. We demonstrated that Ag NPs induced haemolysis. They also increase platelet adhesion without having any impact on platelet aggregation. Finally, they also had procoagulant potential. Bringing all data from these tests together, the no observed effect concentration is 5 μg/mL.

Background Signals of an increased risk of myocardial infarction ( MI ) have been identified with dabigatran etexilate in randomized controlled trials ( RCTs ). Methods and Resules We conducted searches of the published literature and a clinical trials registry maintained by the drug manufacturer. Criteria for inclusion in our meta‐analysis included all RCT s and the availability of outcome data for MI , other cardiovascular events, major bleeding, and all‐cause mortality. Among the 501 unique references identified, 14 RCT s fulfilled the inclusion criteria. Stratification analyses by comparators and doses of dabigatran etexilate were conducted. Peto odds ratio ( OR PETO ) values using the fixed‐effect model ( FEM ) for MI , other cardiovascular events, major bleeding, and all‐cause mortality were 1.34 (95% CI 1.08 to 1.65, P =0.007), 0.93 (95% CI 0.83 to 1.06, P =0.270), 0.88 (95% CI 0.79 to 0.99, P =0.029), and 0.89 (95% CI 0.80 to 1.00, P =0.041). When compared with warfarin, OR PETO values using FEM were 1.41 (95% CI 1.11 to 1.80, P =0.005), 0.94 (95% CI 0.83 to 1.06, P =0.293), 0.85 (95% CI 0.76 to 0.96, P =0.007), and 0.90 (95% CI 0.81 to 1.01, P =0.061), respectively. In RCT s using the 150‐mg BID dosage, the OR PETO values using FEM were 1.45 (95% CI 1.11 to 1.91, P =0.007), 0.95 (95% CI 0.82 to 1.09, P =0.423), 0.92 (95% CI 0.81 to 1.05, P =0.228), and 0.88 (95% CI 0.78 to 1.00, P =0.045), respectively. The results of the 110‐mg BID dosage were mainly driven by the RE ‐ LY trial. Conclusions This meta‐analysis provides evidence that dabigatran etexilate is associated with a significantly increased risk of MI . This increased risk should be considered taking into account the overall benefit in terms of major bleeding and all‐cause mortality.

Proactively managing the risk of marketed drugs: experience with the EMA Pharmacovigilance Risk Assessment Committee

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired disorder of the haematopoietic stem cell that makes blood cells more sensitive to the action of complement. PNH patients experience an increased risk of arterial and venous thrombosis – major causes of death due to this disease. Though many potential interlaced mechanisms are suspected, extracellular vesicles (EVs) of various origins may play a central role. The processes possibly involved are haemolysis, platelet activation, injured endothelial cells and monocyte activation. The impact of transfusion should be evaluated. A better understanding of the mechanisms involved may help to propose guidelines for the prophylaxis and treatment of thrombosis in PNH. In this paper, we propose an updated review of the pathophysiology of the underlying mechanisms of thrombosis associated with PNH, with specific focus on the prominent role of EVs.Keywords: haemoglobinuria; thrombosis; complement; extracellular vesicles; transfusion(Published: 24 March 2014)Citation: Journal of Extracellular Vesicles 2014, 3: 23304 - http://dx.doi.org/10.3402/jev.v3.23304

Several direct oral anticoagulants (DOACs) are now widely used in the prevention and treatment of thromboembolic events. Unlike vitamin K antagonists, DOACs exhibit predictable pharmacokinetics and pharmacodynamics. DOACs are to be administered at fixed doses without routine coagulation monitoring. However, in some patient populations or specific clinical circumstances, measurement of drug exposure may be useful, such as in suspected overdose, in patients with a haemorrhagic or thromboembolic event during treatment with an anticoagulant, in those with acute renal failure, or in patients who require urgent surgery. This article provides practical guidance on laboratory testing of DOACs in routine practice and summarizes the influence of DOACs on commonly used coagulation assays.

In the paper titled “preventive strategies against bleeding due to nonvitamin K antagonist oral anticoagulants,” the authors’ given first and last names were inverted. Here we provide the right authors’ information as shown above.[This corrects the article on p. 616405 in vol. 2014.]

Traditional anticoagulant agents such as vitamin K antagonists (VKAs), unfractionated heparin (UFH), low molecular weight heparins (LMWHs) and fondaparinux have been widely used in the prevention and treatment of thromboembolic diseases. However, these agents are associated with limitations, such as the need for regular coagulation monitoring (VKAs and UFH) or a parenteral route of administration (UFH, LMWHs and fondaparinux). Several non-VKA oral anticoagulants (NOACs) are now widely used in the prevention and treatment of thromboembolic diseases and in stroke prevention in non-valvular atrial fibrillation. Unlike VKAs, NOACs exhibit predictable pharmacokinetics and pharmacodynamics. They are therefore usually given at fixed doses without routine coagulation monitoring. However, in certain patient populations or special clinical circumstances, measurement of drug exposure may be useful, such as in suspected overdose, in patients experiencing a hemorrhagic or thromboembolic event during the treatment’s period, in those with acute renal failure, in patients who require urgent surgery or in case of an invasive procedure. This article aims at providing guidance on laboratory testing of classic anticoagulants and NOACs. Electronic supplementary material The online version of this article (doi:10.1186/1477-9560-12-24) contains supplementary material, which is available to authorized users.

Dabigatran etexilate (DE), rivaroxaban, and apixaban are nonvitamin K antagonist oral anticoagulants (NOACs) that have been compared in clinical trials with existing anticoagulants (warfarin and enoxaparin) in several indications for the prevention and treatment of thrombotic events. All NOACs presented bleeding events despite a careful selection and control of patients. Compared with warfarin, NOACs had a decreased risk of intracranial hemorrhage, and apixaban and DE (110 mg BID) had a decreased risk of major bleeding from any site. Rivaroxaban and DE showed an increased risk of major gastrointestinal bleeding compared with warfarin. Developing strategies to minimize the risk of bleeding is essential, as major bleedings are reported in clinical practice and specific antidotes are currently not available. In this paper, the following preventive approaches are reviewed: improvement of appropriate prescription, identification of modifiable bleeding risk factors, tailoring NOAC’s dose, dealing with a missed dose as well as adhesion to switching, bridging and anesthetic procedures.

Thromboxane A2 (TXA2) is a labile product formed from arachidonic acid by the way of cyclooxygenase and thromboxane synthase. An overproduction of TXA2 has been detected in a series of diseases where this eicosanoid is assumed to contribute to the underlying pathomechanisms by its potent stimulation of platelet aggregation and smooth muscle contraction. Indeed, literature supports participation of TXA2 in several physiopathological mechanisms such as tumour growth and metastasis, pre-eclampsia, asthma, pulmonary hypertension and in the progression of ischaemic injury after coronary artery occlusion. This evidence provides a strong rationale for pursuing TXA2 blocking strategies in drug development. Therefore, TXA2 receptor antagonists, thromboxane synthase inhibitors and drugs which combine both activities have been developed. Of the 28 thromboxane modulator patents reviewed from January 1997 to December 2000, 16 relate to thromboxane receptor antagonists (TXRAs), seven to thromboxane synthase inhibitors ...

Resistance of cancer cells to chemotherapy is a major issue in acute myeloid leukemia (AML) and is usually due to a clonal selection of resistant leukemic cells. Recently, a horizontal transfer of chemoresistance among tumor cells has been reported via extracellular vesicles (EVs). EVs are vesicles ranging from 0.03 to 1µm generated by almost all cell types. They carry cellular components such as nucleic acids, membrane and cytosolic proteins. By interacting with cells and by transferring their content, these vesicles could modify target cells' phenotype. Because of their presence in biological fluids, a lot of studies have tried to use EVs as biomarkers. The aim of this research is to investigate the role of EVs in chemoresistance in AML. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was compared to its multiresistant strain: HL60/AR. The latter overexpresses multidrug resistance-associated protein 1 (MRP1), an efflux pump conferring resistance to anthracyclines. The transfer of this resistance between the two strains is obtained by treating HL60 with EVs generated by HL60/AR. The production of EVs by HL60/AR was confirmed by transmission electron microscopy. EVs from around 100 to 800 nm were highlighted. EVs from HL60/AR were then isolated by ultracentrifugation and their interaction with HL60 was investigated by labeling EVs with a fluorescent lipid membrane linker. The transmission of this fluorescence to HL60 was monitored by flow cytometry and demonstrated a binding or an incorporation of EVs. In order to check if this interaction has an impact on HL60 resistance to daunorubicin, HL60 were treated with EVs, once for 20 hours or four consecutive times for 48 hours each. The resistance to daunorubicin of EVs-treated HL60 was then assessed by a MTT cytotoxicity assay and revealed that EVs increase survival of cells to daunorubicin. The resistant profile between the cells treated once or four times with EVs were somewhat different and highlighted a potential implication of nucleic acids cargo in this resistance transfer. Indeed, nucleic acids transferred by EVs would need more than 20 hours to impact target cells. Afterwards, we investigated the changes in phenotype that could be responsible for this increased resistance of EVs-treated cells. First, the expression of MRP1 was evaluated by a flow cytometry drug retention assay based on the fluorescence exhibited by daunorubicin and consisting in analysis by flow cytometry of the daunorubicin retention of cells with or without the presence of a specific MRP1 inhibitor. This experiment emphasized an expression of MRP1 after EVs treatment. Oxidative stress also plays an important role in daunorubicin cytotoxicity. Thus reactive oxygen species (ROS) generation assays were performed on EVs-treated cells. These cells showed a decreased production of daunorubicin-induced ROS. We finally analyzed the microRNAs content of isolated EVs. This was performed by TaqMan® Human MicroRNA Array on EVs from HL60/AR but also on EVs generated by HL60. This experiment aimed at highlighting the microRNAs cargo potentially involved in this chemoresistance transfer but also at identifying microRNAs differentially expressed between the two strains to serve as biomarker of chemoresistance in vivo. Out of the 380 microRNAs tested, 32 presented a differential expression between HL60 and HL60/AR. Among them, 5 microRNAs presented a more than 8-fold change between the two strains: miR-19b, miR-20a, miR-488, miR-519a and miR-523. miR-519a was the only microRNA more expressed in EVs produced by HL60, compared to EVs from HL60/AR. In conclusion, this study demonstrates the ability of resistant leukemic cells to transfer, at least partially, their chemoresistance via EVs. This could be due to a direct transfer of MRP1 transported by EVs but also to a nucleic acid transfer. In this context, some microRNAs have been highlighted for their high differential expression in EVs between sensitive and chemoresistant cells. These results bring out a potential clinical use of these circulating microRNAs as chemoresistance biomarkers in AML. Disclosures No relevant conflicts of interest to declare.

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances that are involved in several physiological processes but also in pathological conditions such as inflammation. Since ten years now, it is well known that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional nonsteroidal anti-inflammatory drugs (NSAIDs). Observations that COX-1, involved in several homeostatic processes, played a housekeeping role while COX-2 expression was associated with inflammation and other pathologies such as cancer proliferation have led to the development of COX-2 selective inhibitors in order to reduce the classical side-effects, of which gastric irritation is the most common, associated with the use of conventional NSAIDs.

3-Alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides with nitro, amino or acetylamino groups in the 7-position have been synthesized in an attempt to discover new tissue-selective KATP-channel openers. The compounds were tested as putative pancreatic β-cells KATP-channel openers by measuring their inhibitory activity on the insulin releasing process. The influence of the substituent in the 7-position on the acidic character (pKa) and on biological activity is discussed. The nitrobenzene derivatives were biologically active, but less so than the un-derivatized parent pyridothiadiazine dioxides.

Nimesulide, a non-steroidal anti-inflammatory drug and one of a promising class of selective COX-2 inhibitors, has a very interesting therapeutic profile. Unfortunately, it is poorly soluble in water, which leads to important difficulties in the formulation of injectable solutions. This problem can also affect the bioavailability of nimesulide. To increase the aqueous solubility of the drug a nimesulide-L-lysine salt was synthesized in our laboratory; its aqueous solubility was greater than that of nimesulide (solubility in purified water 7.5 mg mL−1, and 0.01 mg mL-1, respectively). The aim of this study was to compare the anti-inflammatory profiles of nimesulide and nimesulide-L-lysine salt in a two-step in-vitro investigation. First, we evaluated the COX-2 selectivity of the drugs by a method using purified COX-1 and COX-2 enzymes. In a second step we evaluated the effects of the drugs on the production of prostaglandin E2 (PGE2) and proteoglycan by chondrocytes from man. The results obtained confirmed the COX-2 selectivity of the two compounds. Nimesulide-L-lysine had the same anti-inflammatory profile as nimesulide on chondrocyte cultures and better water solubility. Nimesulide-L-lysine should, therefore, be used to prepare injectable preparations and should ameliorate bioavailability after oral treatments.

Because torasemide, a loop diuretic, dose-dependently relaxes canine coronary artery precontracted with thromboxane A2 (TxA2), a series of pyridine derivatives have been investigated with the aim of developing original TxA2 receptor antagonists. A binding test showed the affinity (IC50; the dose reducing binding by 50%) of one sulphonylurea derivative (BM 27) and two sulphonylcyanoguanidine derivatives (BM 114 and BM 115) for the TxA2 receptor of washed platelets from man were 161, 1. 75 and 0.54 μM, respectively. The IC50 of torasemide was only 2.69 μM. Their antagonism was confirmed by the capacity of the compounds to prevent the aggregation of platelets, from man, induced by arachidonic acid, the biological precursor of TxA2 (IC50: BM 114, 29.3 μM; BM 115, 12.0 μM; BM 27, 234 μM; torasemide, 350 μM; sulotroban, 12.3 μM). Similar results were obtained with the best compound BM 115 (IC50 11.5 μM) when U-46619, a stable TxA2 agonist, was used as the aggregating agent. This molecule can be regarded as a template for the design of novel non-carboxylic TxA2 receptor antagonists.

Torasemide, a loop diuretic, has been reported to relax dog coronary artery precontracted by thromboxane A2 (TXA2), an endogenous prostanoid involved in cardiovascular and pulmonary diseases. N-cyano-N′-{[4-(3′-methylphenylamino)pyrid-3-yl]sulphonyl}homopiperidinoamidine (BM-144) and N-isopropyl-N′-[5-nitro-2-(3′-methylphenylamino)-benzenesulphonyl]urea (BM-500), chemically related to torasemide, have been examined for their TXA2 antagonism. The affinity (IC50, the concentration resulting in 50% inhibition) of BM-144 and BM-500 for the TXA2 receptor of washed platelets from man was 0.28 and 0.079 μM, respectively. This is better than for sulotroban (IC50 = 0.93 μM) but less than for SQ-29548 (IC50 = 0.021 μM), two TXA2 antagonists used as reference. The aggregation of platelets from man induced by arachidonic acid was prevented by BM-144 (IC50 = 9.0 μM) and by BM-500 (IC50 = 14.2 μM). Similar results were obtained by use of U-46619, a TXA agonist, as aggregating agent (BM-144, IC50 = 12.9 and BM-500, IC50 = 9.9 μM). The contracting effect of U-46619 on rat stomach strip was abolished by BM-144 (IC50 = 1.01 μM) and BM-500 (IC50 = 2–54 μM). Both drugs (BM-144: IC50 = 0.12 and BM-500: IC50 = 0.19 μM) also relaxed rat aorta precontracted by U-46619; both were more potent than sulotroban (IC50 = 1.62 μM). The two torasemide derivatives (100 μM) did not significantly affect the myo-stimulating effect of some prostaglandins (PGE2, PGI2, PGF2α) or aorta contraction elicited by KCl (30 mM). They did not modify rat diuresis after administration of a 30-mg kg−1 dose. In conclusion, BM-144 and BM-500 can be regarded as novel non-carboxylic TXA2 receptor antagonists and offer a novel template for the design of more potent molecules.

A series of new 2-[(arylsulfonyl)imino]-2,3-dihydrothiazolyl derivatives substituted on the heterocyclic N by a phenoxyacetic moiety was prepared according to a Hantzsch's synthesis between N, N'-disubstituted thioureas and chloroacetaldehyde. The regiochemistry of the cyclocondensation reaction was established by high resolution NMR methods. Potential thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptor antagonism was evaluated using human platelet aggregation assays and radioligand binding studies. The results showed that the affinity for the TxA2/PGH2 receptor was strongly dependent on the position of the oxyacetic acid side chain. On the basis of the X-ray crystal analysis of compound 9f, a molecular modelling was undertaken on compounds 3d, 3e, and 3f. Comparison with the 3D structure of sulotroban was discussed.

3-Alkylamino-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide represents a new class of heterocyclic compounds expressing important pharmacological properties. According to the position of the CN double bond in the thiadiazine ring, this heterocyclic ring system may exist under three different tautomeric forms. By means of spectral and X-ray data collected from selected compounds, the most favourable tautomeric form adopted by 3-alkylamino-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides devoid of an alkyl substituent in the 2- or in the 4-position was determined. The present study giving new insights in the geometrical and conformational aspects of pyridothiadiazinedioxides is important considering the pharmacological potentialities of this class of heterocyclic compounds.

Summary Sulfonylcyanoguanidines and sulfonamidonitroethylenes are bioisosteres of hypoglycemic sulfonylureas and were prepared and evaluated for their insulin release potency from rat pancreatic islets. At 25 μM, both bioisosteres of glibenclamide, sulfonylcyanoguanidine 22 and sulfonamidonitroethylene 23 , were as potent as their parent on insulin secretion.

The N-[(4-cycloheptylaminopyrid-3-yl)sulfonyl]-N'-cycloheptyl urea, a neuroprotective agent, and 10 chemically related sulfonyl(thio)ureas were evaluated in the maximal electroshock seizure test in mice. This sulfonylurea, BM 27, and two stucturally related sulfonylthioureas, BM 11 and BM 34, emerged with a 50% effective dose (ED 50 ) of 2.87, 1.72, and 1.19 mg/kg, respectively. Their anticonvulsant profiles were found to be similar to that of phenytoin: active in the maximal electroshock seizure (MES) test and inactive in chemically induced seizures (pentetrazole, strychnine, bicuculline, picrotoxine, N-methyl-D,L-aspartic acid). These compounds exhibited a higher protective index and potency than those of phenytoin. Additional work remains necessary, however, to determine whether BM 27 is of clinical interest.

SummaryApixaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose for the individual patient may be useful in some situations. Moreover, there is a need for clinicians to know whether coagulation assays are influenced by apixaban use. The aim of this study was to determine which coagulation assay could be used to assess the impact of apixaban on haemostasis and provide good laboratory recommendations for the accurate interpretation of haemostasis assays. Apixaban is spiked at concentrations ranging from 5 to 500 ng/ml in platelet-poor plasma. Routinely used or more specific coagulation assays are tested. Results show a concentration dependent prolongation of aPTT, PT and dilute PT. The sensitivity mainly depends on the reagent, but none of these tests is sensitive enough to ensure an accurate estimation of the pharmacodynamic effect of apixaban. FXa chromogenic assays show high sensitivity and a linear correlation depending on the reagent and/or the methodology. Immunological assays and assays acting below the FXa are not influenced by apixaban. In conclusion, PT and/or dilute PT cannot be used to assess apixaban pharmacodynamic properties. More specific and sensitive assays such as chromogenic FXa assays using specific calibrators are required. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake of apixaban and the sampling is mandatory.

Under hypoxia, cancer cells consume glucose and release lactate at a high rate. Lactate was recently documented to be recaptured by oxygenated cancer cells to fuel the TCA cycle and thereby to support tumor growth. Monocarboxylate transporters (MCT) are the main lactate carriers and therefore represent potential therapeutic targets to limit cancer progression. In this study, we have developed and implemented a stepwise in vitro screening procedure on human cancer cells to identify new potent MCT inhibitors. Various 7-substituted carboxycoumarins and quinolinone derivatives were synthesized and pharmacologically evaluated. Most active compounds were obtained using a palladium-catalyzed Buchwald-Hartwig type coupling reaction, which proved to be a quick and efficient method to obtain aminocarboxycoumarin derivatives. Inhibition of lactate flux revealed that the most active compound 19 (IC 50 11 nM) was three log orders more active than the CHC reference compound. Comparison with warfarin, a conventional anticoagulant coumarin, further showed that compound 19 did not influence the prothrombin time which, together with a good in vitro ADME profile, supports the potential of this new family of compounds to act as anticancer drugs through inhibition of lactate flux. © 2013 Elsevier Ltd. All rights reserved.

SummaryPossibilities to monitor rivaroxaban therapy could be useful in certain circumstances. Prothrombin time (PT) or chromogenic anti-Xa assays such as the Biophen Direct Factor Xa Inhibitor® (DiXaI) have been proposed to estimate rivaroxaban concentrations but are mainly based on in vitro studies. The study aim was to compare PT and Biophen DiXaI® measurements with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements in plasma samples from patients treated with Xarelto®. Fifty-two plasma samples were included. PT was performed using Innovin® and Triniclot PT Excel S®. Biophen DiXaI® was performed according to instructions from the manufacturer. The rivaroxaban plasma concentration ranged between 0 and 485 ng/ml as measured by LC-MS/MS. The limits of quantification were 30 ng/ml and 5 ng/ml for Biophen DiXaI® and LC-MS/MS, respectively. The linear correlation between Biophen DiXaI® and LC-MS/ MS analyses was high for all rivaroxaban concentrations (r2 = 0.95). For concentrations ≤100 ng/ml, r2-value was 0.83. The Bland-Altman analysis showed a mean difference of −16 ng/ml (SD: 25 ng/ml). The PT methods did not correlate well with plasma concentrations measured by LC-MS/MS (r2 ≈ 0.60). In conclusion, the important interindividual variability and the poor correlation with LC-MS/MS preclude the use of PT to estimate rivaroxaban concentrations. Thanks to its small inter-individual variability and good agreement with LC-MS/ MS measurements, we recommend the use of Biophen DiXaI® assays to estimate concentrations of rivaroxaban >30 ng/ml. Quantification of low rivaroxaban levels (

Introduction There is still a need for a general test easily implementable and widely available that may be used to screen all patients on DOACs. A recent study has suggested that the dilute Russell Viper Venom Time (DRVV-T) could be used for the monitoring of DOACs but these results have been generated in vitro. The primary objective of this study is to analyse and compare the results obtained with the DRVV Screen and Confirm tests to the plasma drug levels measured by LC-MS/MS. We also aimed at proposing specific cut-off associated with supratherapeutic levels at Ctrough. Finally, a comparison of our results with those obtained with PT for rivaroxaban and aPTT for dabigatran is also provided. Methods Thirty-two rivaroxaban and 31 dabigatran platelet poor plasma samples from real-life patients were included in the study. Dilute Russell's Viper Venom time was measured using STA®-Staclot®DRVV Screen and Confirm reagents. Prothrombin Time and aPTT have been performed with Triniclot PT Excel S® and RecombiPlastin 2G® and with STA®C.K. Prest and SynthasIL®, respectively. The Hemoclot Thrombin Inhibitor® and the Biophen DiXaI® have been performed to estimate plasma concentration of dabigatran and rivaroxaban, respectively. All methodologies were performed on a STA-R Evolution® analyser according with the recommendation of the manufacturer, except for RecombiPlastin 2G® which were performed on an ACL-TOP®. All of these tests have been performed according to the recommendations of the manufacturer. The reference LC-MS/MS measurement of plasma drug concentrations were validated according to FDA Guidelines for Industry for Bioanalytical Method (for rivaroxaban) and to the Validation European Medicines Agency guidelines (for dabigatran). Results The plasma concentrations range from 6 to 426ng/mL for rivaroxaban and from 0 to 386ng/mL for dabigatran as determined by LC-MS/MS. Tables 1 and 2 summarize Spearman correlations and Bland-Atlman analyses for rivaroxaban and dabigatran, respectively. Figures 1 and 2 provide the results of STA®-Staclot®DRVV Screen and Confirm versus LC-MS/MS measurements. Bland-Altman graphs are also provided. Discussion STA®-Staclot®DRVV-Screen and Confirm shows a better correlation than PT or aPTT. Bland-Altman analyses reveal an overestimation of approximately 40ng/mL and large 5th-95th limits of agreement with both STA®-Staclot®DRVV-Screen and Confirm. Specific cut-offs associated with supratherapeutic level (>200ng/mL) at Ctrough have been defined. For STA®-Staclot®DRVV-Screen, results below 125 seconds or below a ratio of 3 could exclude plasma concentrations >200ng/mL for rivaroxaban and dabigatran (Figure 1 - A and B). For STA®-Staclot®DRVV-Confirm, cut-offs must be adapted independently (Figure 2 - A and B). Results below 75 seconds or below a ratio of 2, could exclude rivaroxaban plasma concentrations >200ng/mL. For dabigatran, the threshold could be defined at 90 seconds or at a ratio of 2.5. Conclusion Thanks to its good correlation with plasma drug level, DRVVT can be more informative than PT and aPTT, to exclude supra-therapeutic level of rivaroxaban and dabigatran at Ctrough. However, due to overestimations in plasma drug level, it cannot be recommended to accurately estimate plasma drug concentrations which require more specific coagulation assays or LC-MS/MS measurements. Disclosures: No relevant conflicts of interest to declare.

Introduction: Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required. Objectives: These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/interassay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles. Methods: Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37°C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes and subjected to TGA. Results: The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma. Conclusions: TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients. Keywords: tissue factor; microvesicles; cancer; thrombin generation Access the supplementary material to this article: Refractive index estimation. Please see Supplementary files in the column to the right (under Article Tools online). (Published: 18 March 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 19728 - http://dx.doi.org/10.3402/jev.v2i0.19728

Introduction : Extracellular vesicles (EVs) are shed from cells and carry markers of the parent cells. Vesicles derived from cancer cells reach the bloodstream and locally influence important physiological processes. It has been previously shown that procoagulant vesicles are circulating in patients’ fluids. These EVs are therefore considered as promising biomarkers for the thrombotic risk. Because of their small size, classical methods such as flow cytometry suffer from limitation for their characterisation. Atomic force microscopy (AFM) has been proposed as a promising complementary method for the characterisation of EVs. Objectives : The objectives of this study are: (a) to develop and validate AFM with specific antibodies (anti-TF) and (b) to compare air and liquid modes for EVs’ size and number determination as potential biomarkers of the prothrombotic risk. Methods : AFM multimode nanoscope III was used for air tapping mode (TM). AFM catalyst was used for liquid Peak Force Tapping (PFT) mode. Vesicles are generated according to Davila et al.'s protocol. Substrates are coated with various concentrations of antibodies, thanks to ethanolamine and glutaraldehyde. Results : Vesicles were immobilised on antibody-coated surfaces to select tissue factor (TF)-positive vesicles. The size range of vesicles observed in liquid PFT mode is 6–10 times higher than in air mode. This corresponds to the data found in the literature. Conclusion : We recommend liquid PFT mode to analyse vesicles on 5 µg/ml antibody-coated substrates. Keywords: extracellular vesicles; tissue factor; atomic force microscopy; coagulation; biomarker (Published: 27 August 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 21045 - http://dx.doi.org/10.3402/jev.v2i0.21045 To access the supplementary material to this article, please see Supplementary files under Article Tools online.

The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Introduction Dabigatran etexilate (DE) is an oral direct thrombin inhibitor approved for prevention of stroke and systemic embolism in adult patients with non-valvular atrial fibrillation (NVAF). In the RE-LY trial, myocardial infarction (MI) rates were increased with DE 110mg bid and 150mg bid when compared to warfarin. The risk of MI associated with the use of DE was assessed in a previous meta-analysis of 7 non-inferiority randomized controlled trials (RCTs) showing a significant 33% increase in MI. We performed an updated meta-analysis of RCTs comparing DE with active comparators or placebo to assess the effect of this agent on MI risk as a primary objective. The outcome of major bleeding (MB) and all-cause mortality was also assessed to provide global safety and efficacy measure. Stratifications by comparators (enoxaparin, warfarin or placebo) and by studies using the 150mg bid and the 110mg bid dose regimen were performed. Materials and Methods We conducted searches of the published literature and a clinical-trials registry maintained by the drug manufacturer till 22th of March, 2013. Criteria for inclusion in our meta-analysis included all RCTs and the availability of outcome data for MI, MB and all-cause mortality. Among the 423 unique references identified, 13 RCTs fulfilled the inclusion criteria. All methodologies were performed according to the PRISMA Statement. Results Myocardial infarction occurs in 287 of 23,839 patients (1.20%) treated with DE and in 106 of 13,536 patients treated with controls (0.78%). Major bleeding occur in 948 of 27,063 patients (3.50%) treated with DE and in 551/15,341 patients (3.59%) treated with controls. Death occurs in 989 of 24,162 patients (4.09%) treated with DE and in 570 of 14,498 patients (3.93%) treated with controls. Overall OR for MI, MB and all-cause mortality were 1.32 (95% CI; 1.07-1.63; P=0.010), 0.85 (95% CI: 0.79-0.98; P=0.010) and 0.90 (95% CI; 0.81-1.00; P=0.046) (Figure 1). When compared to warfarin, OR for MI, MB and all-cause mortality were 1.38 (95% CI: 1.08-1.77; P=0.010), 0.85 (95% CI: 0.75-0.95; P=0.006) and 0.90 (95% CI: 0.81-1.01; P=0.069), respectively (Figure 1). In RCTs using the 150mg bid dose regimen, OR for MI, MB and all-cause mortality were 1.44 (95% CI: 1.09-1.90; P=0.010), 0.92 (95% CI: 0.81 to 1.05; P=0.228) and 0.88 (95% CI: 0.78-1.00; P=0.045), respectively (Figure 2). Results of the 110mg bid dose were mainly driven by the RE-LY trial. Discussion DE significantly reduced MB and all-cause mortality compared to controls. However, while the reduction of MB is statistically significant versus warfarin, the reduction in all-cause mortality is not (Figure 1B & 1C). The increased risk of MI with the 150mg bid dose is significant but the reduction in MB and mortality is non-statistically significant (Figure 2). Taken together, these remarks suggest that in frail patients presenting comorbidities, the choice of the 150mg bid dose should be carefully discussed and the 110mg bid dose might be considered. Based on our results, one cannot conclude that the 110mg bid dose is associated with a higher risk of MI. However, in terms of absolute risk, such an increased risk of MI should be tempered when compared to the outcomes of stroke or systemic embolism, MB and all-cause mortality. The results from the RE-LY trial showed that the benefits of DE over warfarin outweigh this increase risk of MI. The risk difference was greatly in favor of DE regarding the composite of stroke/systemic embolism, MI, MB and all-cause mortality. Conclusion This meta-analysis of RCTs provides robust evidence that DE is associated with an overall significant 32% increase in the risk of MI. The risk was principally identified when warfarin is used as comparator (38% increase). In RCTs using the 150mg bid DE dose, a significant 44% overall increased risk of MI was identified. No definitive conclusion about the absence of the risk of MI with the 110mg bid DE dose can be drawn at this time. However, this increase risk has to be tempered with the overall benefit of DE especially in the patients with NVAF. In conclusion, we suggest that health care professionals and regulators should consider additional risk minimization strategy to prevent the risk of MI in vulnerable population. Disclosures: No relevant conflicts of interest to declare.

New series of original 2-aryloxy/arylamino-5-cyanobenzenesulfonylureas were synthesized and evaluated as thromboxane A2 receptor (TP receptor) antagonists. A functional pharmacological test was used, which consisted of measuring the inhibition of intracellular calcium mobilization in a model of mammalian cell line that specifically over-expressed the individual TPα or TPβ isoforms. 2-Arylamino-5-cyanobenzenesulfonylureas exhibited virtually identical affinity and/or functional activity than 2-aryloxy-5-cyanobenzenesulfonylureas for both TPα and TPβ, but some 2-aryloxy-substituted compounds showed increased selectivity for TPβ relative to TPα. Several compounds were found to be as potent as the 2-arylamino-5-nitrobenzenesulfonylurea reference compound BM-573, supporting the view that the bioisosteric replacement of the nitro group by a cyano group was tolerated. TP receptor antagonist activity of the most promising molecules was confirmed in a platelet aggregation assay using the TP receptor agonist U-46619 as a proaggregant. Three compounds (7e, 7h and 8h) were identified as leads for further non-clinical pharmacological and toxicological studies.

Ischemia-reperfusion disturbs endothelial physiology and generates a proinflammatory state. Animal studies showed that clonidine administered prior hypoxia improves posthypoxic endothelial function. To investigate this effect in human, we have assessed the postischemic endothelium function and the proinflammatory state in healthy volunteers with and without clonidine. Seven volunteers were included. Each subject underwent the experimental protocol (15 minutes nondominant forearm ischemia) with and without clonidine. Endothelial function was assessed by flow-mediated dilatation (FMD) in the brachial artery before ischemia (FMDPI), immediately after ischemia (FMDIAI), and 15 minutes after ischemia (FMD15AI). Neutrophil (CD11b/CD18) and platelet (CD42b) activations were measured by flow cytometry during reperfusion in blood samples from ischemic (local) and nonischemic (systemic) forearms. Proinflammatory state was assessed by serum concentration of interleukin (IL)-1β and -6. Clonidine does not influence baseline FMD (P = 0.118) but improves FMDIAI (P = 0.018) and FMD15AI (P = 0.018). It increases platelet activation in systemic circulation (P = 0.003) during reperfusion but not in local circulation (P = 0.086). Clonidine increases neutrophil activation in local circulation (P = 0.001) but not in systemic circulation (P = 0.642). In local circulation, clonidine decreases IL-6 (P = 0.044) but does not influence IL-1β (P = 0.113). By contrast, it decreases both IL-6 (P = 0.026) and IL-1β (P = 0.027) concentrations in systemic circulation. In conclusion, clonidine improves endothelial function and modulates inflammation during reperfusion.

Introduction: Rivaroxaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations. Aim: To determine which coagulation assay could be used to assess the impact of rivaroxaban on haemostasis and provide guidelines for the interpretation of routine lab tests. Materials: Rivaroxaban was spiked at concentrations ranging from 11 to 1090 ng/mL in plateletpoor plasma. A large panel of coagulation assays was tested. Results: A concentration dependent prolongation of aPTT, PT, dPT, PiCT was observed. PT and dPT were the most sensitive chronometric assays but results varied depending on the reagent (Triniclot PT Excel S > Recombiplastin 2 G > Neoplastin R > Neoplastin CI + > Triniclot PT Excel > Triniclot PT HTF > Innovin). FXa chromogenic assays showed the highest sensitivity. In TGA, Cmax was the most sensitive parameter with the tissue factor induced pathway. Rivaroxaban interferes on haemostasis diagnostic tests such the measurement of clotting factors, fibrinogen, antithrombin, proteins C and S, activated protein-C resistance and Xa-based chomogenic assays. Conclusions: PT may be used as screening test to assess the risk of bleedings. A more specific and sensitive assay such as Biophen DiXaI using calibrators should be used to confirm the concentration of rivaroxaban. We also propose cut-off associated with a bleeding or thrombosis risk based on pharmacokinetic studies. Standardization of the time between the last intake of rivaroxaban and the sampling is mandatory.

Background: Silicon carbide (SiC) presents noteworthy properties as a material such as high hardness, thermal stability, and photoluminescent properties as a nanocrystal. However, there are very few studies in regard to the toxicological potential of SiC NPs. Objectives: To study the toxicity and biodistribution of silicon carbide (SiC) nanoparticles in an in vivo rat model after acute (24 h) and subacute (28 days) oral administrations. The acute doses were 0.5, 5, 50, 300 and 600 mg·kg{sup −1}, while the subacute doses were 0.5 and 50 mg·kg{sup −1}. Results: SiC biodistribution and elemental composition of feces and organs (liver, kidneys, and spleen) have been studied by Particle-Induced X-ray Emission (PIXE). SiC and other elements in feces excretion increased by the end of the subacute assessment. SiC did not accumulate in organs but some elemental composition modifications were observed after the acute assessment. Histopathological sections from organs (stomach, intestines, liver, and kidneys) indicate the absence of damage at all applied doses, in both assessments. A decrease in the concentration of urea in blood was found in the 50 mg·kg{sup −1} group from the subacute assessment. No alterations in the urine parameters (sodium, potassium, osmolarity) were found. Conclusion: This is the first studymore » that assesses the toxicity, biodistribution, and composition changes in feces and organs of SiC nanoparticles in an in vivo rat model. SiC was excreted mostly in feces and low traces were retrieved in urine, indicating that SiC can cross the intestinal barrier. No sign of toxicity was however found after oral administration. -- Highlights: ► SiC nanoparticles were orally administered to rats in acute and subacute doses. ► SiC was found in low traces in urine. It is mostly excreted in feces within 5 days. ► SiC excretion rate, feces and organ elemental composition change with time. ► No morphological alteration were found on GI tract, liver, kidneys, or spleen. ► Urea increased in blood in the subacute assessment. No change in urine properties.« less

Introduction: Ponatinib is a BCR-ABL tyrosine kinase inhibitor (TKI) indicated in adult patients suffering from chronic myeloid leukemia (CML) or “Philadelphia-chromosome positive” (Ph+) acute lymphoblastic leukemia resistant or intolerant to dasatinib or nilotinib. A high incidence of arterial events including peripheral artery occlusive diseases has been observed with ponatinib during the clinical development. A similar risk was also suspected with nilotinib, another BCR-ABL TKI used in CML. To date, no signal of increased risk of vascular thrombosis and embolism was found in clinical trials with imatinib and dasatinib. However, some cases were reported post-marketing. Aim: To perform a meta-analysis to assess the risk of vascular occlusive events (VOE) with novel generation BCR-ABL TKI (i.e. bosutinib, dasatinib, nilotinib and ponatinib) in CML patients compared with standard of care (i.e. imatinib). Stratifications by BCR-ABL TKI are performed to provide product specific risk assessment. Data on the overall survival and the major molecular response were also extracted to provide global benefit-risk evaluation. Methods: Study selection was performed in two stages. Initially, two independent reviewers screened abstracts and titles against inclusion and exclusion criteria (see protocol at PROSPERO 2014:CRD42014014147). Then, both reviewers assessed potentially relevant papers in their entirety, deciding on inclusion. Studies included all randomized controlled trials (RCT), comparing BCR-ABL TKI versus the reference therapy. Data on major molecular response, overall survival and VOE were extracted. The meta-analysis was performed using a fixed-effect model. Odds ratios (OR) with 95% confidence intervals (CI) were computed using the Peto method. Statistical heterogeneity across the various trials was tested using the Cochran's Q statistic and quantified by the I2 value. Funnel plot asymmetry was assessed by the method of Egger's linear regression, the Begg and Mazumbar rank correlation and the Owrin's fail safe N tests. Results: Among the 10 studies included, 3 studies did not report overall survival and 9 studies reported VOE and the major molecular response. Risk of VOE was increased with dasatinib (OR 3.86; 95%CI: 1.33 to 11.18), nilotinib (OR: 3.45; 95%CI: 2.07 to 5.63) and ponatinib (OR: 3.47; 95%CI: 1.23 to 9.78) compared to imatinib in patients with Ph+ CML (Figure 1). No significant difference was found with bosutinib (OR: 2.77; 95%CI: 0.39 to 19.77) probably due to a lack of power, but a trend was also demonstrated (Figure 1). Novel generation TKI increased the rate of major molecular response at 1 year compared to imatinib (overall OR: 2.22; 95%CI: 1.87 to 2.63) (Figure 2). No statistical difference in term of overall survival at one year was found (overall OR: 1.23; 95%CI: 0.67 to 2.23) (Figure 3). Unfortunately, the inaccessibility to individual data and to the time-to-event along with the fact that the evaluation criteria were not similar between studies can introduce a bias in the analysis. However, neither heterogeneity nor publication bias were found, supporting the robustness of our results. The risk of VOE is likely to be dose-related; however, currently available data on dose-efficacy and dose-toxicity relationship are not sufficient to make a formal recommendation on dose reduction, since there is a risk that lower doses might have reduced efficacy. However, safety and efficacy data concerning dose reduction following major cytogenetic response have been included in the EU-SmPC of Iclusig¨ (ponatinib) as a risk minimization measure. Conclusion: Dasatinib, nilotinib and ponatinib increase VOE. Novel generation TKI improve major molecular response but not the overall survival at one year. Further dose-ranging studies are required to define the dose regimen of each BCR-ABL TKI that will provide the best benefit-risk profile. Monitoring the response at the individual level might be of utmost importance to reduce the risk of VOE while keeping the benefit in term of major molecular response. Figure 1. Forest plot of the risk of VOE in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Figure 1. Forest plot of the risk of VOE in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Figure 2. Forest plot of the major molecular response in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Figure 2. Forest plot of the major molecular response in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Figure 3. Forest plot of the overall survival in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Figure 3. Forest plot of the overall survival in patients with Ph+ leukemia treated with new generation TKIs versus imatinib. Disclosures Graux: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.