Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

[Pharmacologic heterogeneity of new anticoagulants]. / Hétérogénéité pharmacologique des nouveaux anticoagulants.

Amongst numerous promising anticoagulant molecules, rivaroxaban (Xarelto(®)), dabigatran (Pradaxa(®)) and apixaban (Eliquis(®)) have been registered outside the USA in the prevention of thromboembolic events in patients undergoing total hip or knee prosthetic replacement. Rivaroxaban however has been granted authorisation by the FDA for the thromboprophylaxis after surgery for total hip or knee surgery. Dabigatran has been granted authorisation by the FDA in non-valvular atrial fibrillation (RE-LY trial) while rivaroxaban is expecting approval in this same indication (ROCKET trial). Phase III results in the treatment and in the secondary prevention of established venous thrombosis and pulmonary embolism are encouraging. These small molecules are obtained by chemical synthesis, their molecular weight is lower than 500 daltons. Many coagulation tests may be affected by these molecules. Those modifications should be known in order to avoid misinterpretation of the tests but could also be used to measure plasma concentrations of these products. The choice of a non specific global and readily available test has been documented (Quick time for rivaroxaban and aPTT for dabigatran). Anti-Xa (for rivaroxaban) and anti-IIa (for dabigatran) activities should however be preferred, expressed in ng/ml with calibrated plasmas (containing predetermined concentration of the tested drug). The half-life is around 8 to 12 hours, with a peak activity 2 to 4 hours after ingestion. Dabigatran is mainly eliminated via the kidney, hence requiring dose-adjustment in case of moderate renal insufficiency, and contra-indicated in case of severe renal insufficiency. Rivaroxaban being excreted via kidney and liver, some precautions should apply in case of liver insufficiency. No data are available in pregnancy or pediatrics, clinical trials are ongoing. There are few interactions with concomitant drugs, which should not be ignored. The short half-life of these new agents compensates for the lack of any specific antidote in many instances. Their oral administration, without the need for dose adjustment, and without requirement for a laboratory monitoring will increase their use in a large number of patients, in those indications for which an approval has been granted by health authorities.

Oral glycoprotein IIb/IIIa antagonism in patients with coronary artery disease.

Dose-finding studies and trials of interaction of oral glycoprotein IIb/IIIa antagonists with other antiplatelet agents have been limited. We hypothesized that these detailed assessments could be first performed in patients with stable coronary artery disease (CAD) and then extrapolated to the target population. To this end, we performed 2 sequential studies. The first study examined the dose-related effects on indexes of platelet and vascular function induced by the oral inhibitor RPR 109891, when given alone and in combination with aspirin, in patients (n = 100) with stable CAD. The second study (the Antagonism of the FIbrinogen Receptor after Myocardial Events trial) assessed the pharmacodynamics and safety of derived regimens in patients (n = 320) with unstable coronary syndromes. In patients with stable CAD, platelet aggregation was dose dependently inhibited by RPR 109891, and the dose-response relation was shifted to the right by the concomitant administration of aspirin (p = 0.0001). The degree of platelet inhibition induced by 3 doses of RPR 109891 (plus aspirin) was lower in patients with unstable than stable CAD. No drug-related major bleeding occurred in either study. RPR 109891 treatment was associated with acute and delayed thrombocytopenia. In conclusion, chronic treatment with an oral glycoprotein IIb/IIIa antagonist (1) induces antiplatelet effects that are potentiated by concomitant administration of aspirin, (2) may require dose adjustment in syndromes of platelet activation, (3) is associated with a low rate of clinically significant bleeding when doses inducing incomplete inhibition of platelet aggregation are used, and (4) requires frequent monitoring of platelet count unless reliable predictors of delayed thrombocytopenia become available.

Are low-molecular-weight heparins useful for the prophylaxis and treatment of arterial thrombi?

The pharmacologic specificity of low-molecular-weight heparins (LMWHs) has enabled multiple attractive developments in the prophylaxis and treatment of arterial thrombosis. Their high antithrombotic potency associated with a potentially lower induced bleeding risk, the lack of platelet interaction, the prevention of myointimal hyperplasia, and the lower incidence of heparin-induced thrombocytopenia, are major advantages. New studies in cardiology and vascular surgery demonstrate a high efficacy for LMWHs associated with a low risk.

A randomised controlled trial of a low-molecular-weight heparin (Enoxaparin) to prevent deep-vein thrombosis in patients undergoing vascular surgery.

The incidence of postoperative deep vein thrombosis (PDVT) after aortic surgery and lower limb revascularisation has not been assessed by a large prospective study. In a prospective randomised trial the effect of a low-molecular-weight heparin fragment, Enoxaparin (ENX) 4200 anti factor Xa IU once daily was compared to that of unfractionated heparin (UFH) 7500 IU twice daily. Two hundred and thirty-three consecutive patients were classified into three groups, aortic or aortoiliac and aneurysmectomy (n = 75), aorto-femoral bypass for atherosclerotic disease (n = 71), and femoropopliteal or femorodistal bypass (n = 87). Patients were analysed for development of deep vein thrombosis by Duplex scanning and, if positive, by venography between the seventh and tenth postoperative day. PDVT was present in 10 patients in the ENX group and in four patients in the UFH group (8.2 and 3.6% respectively, NS). The incidence of PDVT was 8% after aortic or aortoiliac aneurysmectomy, 7% after aortofemoral revascularisation, and 3.4% after femoropopliteal or femorodistal bypass. The overall incidence of PDVT after aortic surgery was 7.5% (95% CI 5.4-9.7). There was no pulmonary embolism. Intra-operative blood loss and postoperative bleeding events did not differ significantly between the ENX and UFH groups. After 1 month follow-up, no clinical event or death could be related to PDVT or pulmonary embolism. In conclusion, in vascular surgery ENX is as safe and effective in the prevention of PDVT as is UFH.

Subcutaneous low-molecular-weight heparin compared with continuous intravenous unfractionated heparin in the treatment of proximal deep vein thrombosis.

BACKGROUND: A low-molecular-weight heparin, enoxaparin sodium, has been shown to be effective and safe in preventing deep vein thrombosis both in general surgery and in high-risk orthopedic surgery. We conducted a controlled, randomized trial with enoxaparin in the treatment of established deep vein thrombosis. METHODS: In a multicenter trial, we compared fixed-dose subcutaneous enoxaparin, given twice daily, with adjusted-dose intravenous unfractionated heparin (UFH) given by continuous intravenous infusion for the initial 10 days of treatment of patients with proximal vein thrombosis. The primary efficacy outcome was the change of the size of the thrombus assessed by repeated venograms between day 0 and day 10. The primary analysis of safety was based on the incidence of major bleeding during 10 days of treatment. RESULTS: There were 67 patients in each group. Venographic assessment of clot size evolution between day 0 and day 10 showed a statistically significant superiority (P < .002) of enoxaparin over the reference treatment with UFH. Moreover, the incidence of overall recurrent thromboembolic events during 10 days of treatment was significantly higher (P < .002) in the UFH group (seven of 67) than in the enoxaparin group (one of 67). There were no serious bleeding complications in either group. CONCLUSIONS: Enoxaparin is at least as effective and safe as UFH under the conditions of this study. Moreover, it is more comfortable for patients and less time-consuming for nurses and laboratories. Thus, our study confirmed, with the use of enoxaparin, other observations that low-molecular-weight heparin provides a real therapeutic advance in the treatment of deep vein thrombosis.

Anti Xa activity and prothrombinase inhibition in patients treated with two different doses of enoxaparin in gynecologic surgery.

A prospective open study was performed in a series of 547 patients undergoing gynecologic surgery. A dose of 20 mg of enoxaparin was administered to all patients 2 hours before surgery. Then patients at high risk of thrombosis (mostly oncologic surgery) received 40 mg of enoxaparin daily whereas those at moderate risk received 20 mg of enoxaparin daily. The principal aim of this prospective open study was to monitor amidolytic anti Xa activity and to study the inhibition of intrinsic prothrombinase using prothrombin consumption measurement as a simple and global test. A second aim was to investigate efficacy and tolerance of these regimens. Treatment tolerance was satisfactory with both regimens since the total incidence of bleeding was 1.8%. A single patient developed a clinically significant thrombosis during hospital stay. The results confirm and extend previous reports regarding a dose effect relationship between the dose of Enoxaparin and plasma Anti Xa activity 3 hours after s.c. injection. A significant relationship was found between Anti Xa activity and patients body weight. Interestingly a dose dependent inhibition of intrinsic prothrombinase was observed when using a one stage prothrombin consumption assay in whole blood. This test in whole blood can be considered as closer to physiological conditions than assays performed in citrated platelet rich or platelet poor plasma samples. The mechanism of intrinsic prothrombinase inhibition during prophylactic treatment with enoxaparin requires further investigation.

[Peri- and postoperative use of low molecular weight heparin in peripheral vascular surgery]. / Utilisation per et postopératoire d'une héparine de bas poids moléculaire en chirurgie vasculaire périphérique.

A pilot study has been conducted in ten consecutive patients undergoing femoro-popliteal reconstruction or distal vascular surgery under epidural anaesthesia. Immediately before arterial cross-clamping, enoxaparin (E) (75 anti-Xa IU.kg-1) was injected intravenously (i.v.). During surgery, washing of the saphenous or polytetrafluoroethylene (PTFE) graft has been performed using enoxaparin. Enoxaparin (75 anti-Xa IU.kg-1) was administered subcutaneously (S.C.) 8 hours after the i.v. injection, and then every 12 hours during 10 days. The patency of the vascular reconstruction and the side-effects of E administration were evaluated clinically before and during surgery, then by a daily clinical examination. Echo-Doppler and/or arteriography were also performed preoperatively and on the 10th postoperative day. Haematocrit, platelet count, activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen and anti-Xa activity were assessed. None of the patients developed venous or arterial thrombosis and all the by-pass grafts remained patient. Only one minor surgical bleeding occurred on the first post operative day, despite anti-Xa levels in the expected range. One patient developed minor haematomas at the injection site. No bleeding was observed. Further randomized studies comparing LMWH and UH are required in order to substantiate these preliminary clinical and biological findings.

Prevention of thromboembolic disease in general surgery with enoxaparin (Clexane).

Different low molecular weight heparins have been compared with unfractionated heparin for the prevention of venous thrombosis in general surgery in about 20 controlled therapeutic trials since 1984. The Multicentre Genox Study* included 892 patients in three consecutive randomized studies with a daily dose of 60, 40 and 20 mg of enoxaparin, respectively, the first injection given 2 h prior to surgery, as compared to subcutaneous unfractionated heparin 5000 IU tds. All included patients had at least one additional risk factor for thrombosis, in addition to surgical risk and to age over 40 years. This trial was the first to compare low molecular weight heparins to controls receiving three daily injections of unfractionated heparin instead of two, and to use three different daily doses of a low molecular weight heparin. The trials were conducted in abdominal, gynaecologic, urologic and thoracic surgery. About 30% of patients had malignancy. Percentages of positive 125I-fibrinogen uptake test (isotopic thrombosis) were comparable: 2.9, 2.8 and 3.8% with enoxaparin and 3.8%, 2.7% and 7.6% with unfractionated heparin, and were not significantly different. There was a significant decrease in haematocrit and haemoglobin only in those patients receiving 60 mg of enoxaparin. The conclusion of this study was that a daily dose of 20 or 40 mg of enoxaparin (approximately 2000 and 4000 anti-Factor Xa IU) was safe and not statistically different from unfractionated heparin. No biological control, except platelet counts before treatment and then twice a week, is required in prophylaxis.

Results of thrombolysis in the treatment of arterial ischemia of the limbs according to mode of administration.

After reviewing the principles, results and complications of thrombolytic therapy with "classical" agents (Streptokinase and Urokinase) used via intravenous, intraarterial route, or intraoperatively, and with more "modern" agents (APSAC, scuPA, tPA), we discuss the future of thrombolysis in the treatment of arterial ischemia of the limbs. Several items need to be clarified: --indication of thrombolysis among other treatments, mainly surgery, of arterial ischemia depends on the clinical staging of ischemia, its causes and the site of arterial obstruction; --method of delivery of the thrombolytic agent must provide the highest local concentration and the lowest systemic side effects; --efficacy of each thrombolytic agent must be analyzed when used in peripheral arterial ischemia, but also in other diseases such as myocardial infarction.

[Arterial thrombolysis. Review and future prospects]. / Thrombolyse artérielle. Bilan et perspectives d'avenir.

After describing the mechanism of action, results and local and general complications of classical thrombolytic agents (streptokinase and urokinase) administered systemically, locally and peroperatively, then of modern thrombolytic agents (acyl enzyme, tPA and scuPA), the future perspectives of arterial fibrinolysis are discussed. These are based upon: analysis of the comparative efficacy of the different thrombolytic agents in the light of results seen, not only in peripheral arterial pathology but also in other indications, as in coronary pathology; discussion of the methods of administration of the thrombolytic agent in such a way as to obtain an optimal local concentration and at the same time reduced systemic fibrinolysis. better definition of the indications of thrombolysis in acute ischemia of the limbs in comparison with other therapeutic approaches, in particular surgery, taking into account the degree of ischemia, of etiological mechanism and the site of the arterial obstruction.