EP217609, a neutralisable dual-action FIIa/FXa anticoagulant, with antithrombotic effects in arterial thrombosis

2015 ◽  
Vol 113 (02) ◽  
pp. 385-395 ◽  
Author(s):  
Ghina Alame ◽  
Pierre H. Mangin ◽  
Monique Freund ◽  
Nadia Riehl ◽  
Stéphanie Magnenat ◽  
...  
Keyword(s):  
Blood Loss ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Parent Compound ◽  
Thrombin Inhibitor ◽  
Potential Candidate ◽  
Low Doses ◽  
Response Curves ◽  
Dual Action

SummaryEP217609 is a new synthetic parenteral dual-action anticoagulant combining a direct thrombin inhibitor (α-NAPAP analog), an indirect factor Xa inhibitor (fondaparinux analog) and a biotin moiety allowing its neutralisation. EP217609 exhibited similar in vitro anticoagulant properties as its parent compounds. On the basis of dose-response curves, we identified low and moderate doses of EP217609 resulting in similar ex vivo prolongation of the APTT as α-NAPAP analog and comparable ex vivo anti-FXa activity as fondaparinux. The effects of EP217609 were compared to those of its parent compounds used alone or in combination in two models of experimental thrombosis induced by FeCl3 injury of the carotid artery or mechanical injury of atherosclerotic plaques in ApoE-deficient mice. When administered at low doses increasing the APTT by only 1.1 fold, EP217609 significantly reduced the thrombus area in both models as compared to α-NAPAP analog or fondaparinux alone, but not to the combination of these drugs. In contrast, at higher doses increasing the APTT 1.5 times, EP217609 was not superior to either parent compound. Low doses of EP217609 did not prolong the tail bleeding time or increase the volume of blood loss, although a tendency towards an increased blood loss was observed in five out of 12 mice. Finally, the effects of EP217609 could be neutralised in vivo by injection of avidin. The pharmacological profile of EP217609, its performance in arterial thrombosis models and its possible neutralisation make it an interesting molecule and a potential candidate as an antithrombotic drug.

scholarly journals ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

2018 ◽  
Vol 10 (4) ◽  
pp. 44
Author(s):  
Mihir K Patel ◽  
Kiranj K. Chaudagar ◽  
Anita A. Mehta
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Platelet Activity ◽  
Aggregation Assay ◽  
Thrombosis Model ◽  
Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

scholarly journals Tranexamic Acid Increases Early Postoperative Pain and Decreases Time to First Opioid Following Total Knee Arthroplasty

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Sachin Seetharam ◽  
Sydney Keller ◽  
Mary Ziemba-Davis ◽  
R. Michael Meneghini MD
Keyword(s):  
Total Knee Arthroplasty ◽  
Postoperative Pain ◽  
Blood Loss ◽  
Tranexamic Acid ◽  
Knee Arthroplasty ◽  
Ex Vivo ◽  
Pain Scores ◽  
Average Pain ◽  
Total Knee

Background and Hypothesis: Tranexamic acid (TXA) decreases blood loss in total knee arthroplasty (TKA). However, TXA evoked pain in rats by inhibiting GABA and glycine receptors in the spinal dorsal horn, and caused cellular death in ex vivo and in vitro human periarticular tissues exposed to clinical concentrations of TXA. We evaluated inpatient postoperative pain and blood loss in TKA performed with and without TXA. Project Methods: 105 consecutive cemented TKAs without TXA were compared to 72 consecutive cemented TKAs with TXA. Procedures were performed by a single surgeon using identical perioperative medical and pain-control protocols. Outcomes included: average of q2-4 hour pain scores during the first 24 hours after PACU discharge, average pain during remainder of stay, final pain score prior to discharge, time in minutes to first opioid after PACU discharge, total opioids in morphine equivalents (MEQs) during the first 24 hours after PACU discharge, average MEQs per remaining days of stay, and mean g/dL pre- to postoperative decrease in hemoglobin. Multivariate analyses accounted for 15 demographics and covariates. Results: The sex (p=0.393), age (p=0.784), and BMI (p=0.930) of the two cohorts were similar. Mean pain during the first 24 hours was greater (4.1 vs. 3.2, p=0.001), MEQs consumed during the first 24 hours were greater (45 vs. 37, p=0.069), and time to first opioid medication was shorter (326 vs. 414, p=0.023) in patients who received TXA. The decrease in hemoglobin was less in patients who received TXA (-2.2 vs. -2.7, p<0.001).   Conclusion and Potential Impact: Our hypothesis based on animal and laboratory studies that TXA may increase early postoperative pain was confirmed by three metrics. Consistent with the effective life of TXA, pain and opioid consumption after 24 hours did not differ based on TXA use. Further work is warranted to investigate the nature consequences associated with TXA, relative to its demonstrated benefits for blood conservation.  

A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2985-2988 ◽  
Author(s):  
Claudine Graf ◽  
Florian Heidel ◽  
Stefan Tenzer ◽  
Markus P. Radsak ◽  
Fian K. Solem ◽  
...  
Keyword(s):  
T Cells ◽  
Tandem Duplication ◽  
Ex Vivo ◽  
Hla Class I ◽  
Protein Domain ◽  
Target Antigen ◽  
Potential Candidate ◽  
Internal Tandem Duplication ◽  
Candidate Target

Abstract The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I–restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results demonstrate that AML leukemic blasts can in principle process and present immunogenic FLT3-ITD neoepitopes. Therefore, FLT3-ITD represents a potential candidate target antigen for the immunotherapy of AML.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

Neutralization of Enoxaparine-lnduced Bleeding by Protamine Sulfate

1990 ◽  
Vol 63 (02) ◽  
pp. 271-274 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
L Cai ◽  
F A Ofosu ◽  
J Hirsh ◽  
M R Buchanan
Keyword(s):  
Blood Loss ◽  
Unfractionated Heparin ◽  
Ex Vivo ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Low Molecular Weight Heparins ◽  
Before And After ◽  
Made In

SummaryIt has been suggested that protamine sulfate is a poor antidote for the bleeding side-effeets of low molecular weight heparins (LMWHs) in vivo, since protamine sulfate does not completely neutralize the anti-factor Xa activity of LMWHs in vitro or ex vivo. Therefore, we performed experiments to compare directly the abilities of protamine sulfate to neutralize the anticoagulant activities of the LMWH, enoxaparine, and unfractionated heparin ex vivo, with its ability to neutralize the bleeding side-effeets of both compounds in vivo. Bleeding was measured as the amount of blood lost from 5 cuts made in rabbits ears before and after treatment with enoxaparine or unfractionated heparin ± protamine sulfate. Plasma anti-factor Xa and anti-thrombin activities ex vivo, were measured chromogenically. Doses of 400 and 1,500 anti-factor Xa U/kg of heparin and enoxaparine, respectively, were required to enhance blood loss to the same extent. Protamine sulfate completely neutralized blood loss induced by both compounds, but did not neutralize the anti-factor Xa nor antithrombin activities ex vivo. We conclude that protamine sulfate is an effective antidote for the bleeding side-effeets of enoxaparine and unfractionated heparin, despite its inability to completely neutralize their anticoagulant activities.

Experimental Pharmacology of Hirunorm: a Novel Synthetic Peptide Thrombin Inhibitor

1996 ◽  
Vol 76 (03) ◽  
pp. 384-392 ◽  
Author(s):  
Rocco Cirillo ◽  
Annalisa Lippi ◽  
Alessandro Subissi ◽  
Giancarlo Agnelli ◽  
Marco Criscuoli
Keyword(s):  
Venous Thrombosis ◽  
Arterial Thrombosis ◽  
Coronary Thrombosis ◽  
Vena Cava ◽  
Thrombin Inhibitor ◽  
Similar Species ◽  
Thrombin Activity ◽  
Experimental Pharmacology ◽  
Anaesthetized Rats

SummaryEnhanced thrombin activity has been associated with coronary thrombosis and with acute and long-term complications following coronary balloon angioplasty. Blocking thrombin activity with specific inhibitors is proposed as a promising antithrombotic therapy. We describe the anticoagulant and antithrombotic properties of hirunorm, a novel synthetic 26-aminoacid peptide thrombin inhibitor, in comparison with r-hirudin and hirulog-1. Hirunorm was equipotent to hirulog-1 and 1/30 as potent as r-hirudin in blocking a-thrombin amidolytic activity (IC50 = 10 ± 2,15 ± 1 and 0.3 ± 0.1 nM, respectively), but it did not affect trypsin, plasmin and t-PA activities at 10 μM. All the compounds inhibited clot-bound thrombin to clots prepared by thrombin hydrolysis of purified fibrinogen in buffer. Hirunorm and hirulog-1 showed similar species-dependent potency in doubling basal in vitro clotting times of human, rat and rabbit plasma (EC200 varied 70 to 200 nM for TT, 0.7 to 16 μM for aPTT and 0.8 to 17 μM for PT), while r-hirudin was always at least three times more active. When assayed by HPLC or by bioassay of the intact peptide, hirunorm was stable against a-thrombin and plasma hydrolases, but it was catabolized by rat liver and kidney enzymes. Venous thrombosis was produced in anaesthetized rats by vena cava ligation following a procoagulant serum injection. Intravenous and subcutaneous hirunorm inhibited venous thrombosis at doses (≤0.3 mg/kg) two-three times higher than those of r-hirudin. Hirulog-1 was as active as hirunorm only after i. v. infusion. Arterial thrombosis was obtained in the anaesthetized rat by chemical (FeCl2) stimulation of a common carotid and i.v. infused hirunorm (1-3 mg/kg/30 min) inhibited it dose-dependently; r-hirudin was partly active only at 3 mg/kg, but hirulog-1 was inactive at either dose. Full antithrombotic doses of hirunorm did not affect the bleeding time as measured from punctured mesenteric vessels, in anaesthetized rats. In conclusion, hirunorm is a potent peptide thrombin inhibitor endowed with antithrombotic activity in models of venous and arterial thrombosis.

Development of Mucoadhesive Buccal Films of Glipizide

1970 ◽  
Vol 1 (2) ◽  
pp. 177-183
Author(s):  
Mona Semalty ◽  
Ajay Semalty ◽  
Ganesh Kumar ◽  
Vijay Juyal
Keyword(s):  
Controlled Release ◽  
Residence Time ◽  
Ex Vivo ◽  
In Vitro Release ◽  
Content Uniformity ◽  
Potential Candidate ◽  
Sodium Carboxymethylcellulose ◽  
Casting Technique ◽  
Buccal Films

For improving bioavailability in controlled release fashion and to circumvent the hepatic first pass effect of glipizide mucoadhesive buccal films of glipizide were prepared by solvent casting technique. Buccal films were prepared using hydroxy propylmethylcellulose, sodium carboxymethylcellulose, carbopol-934P and Eudragit RL-100. Films were evaluated for their weight, thickness, surface pH, swelling index,       in vitro residence time, folding endurance, in vitro release, ex vivo permeation studies and drug content uniformity. The films exhibited controlled release over more than 6 h. From the study it was concluded that the films containing 5 mg glipizide in 4.9 % w/v hydroxy propylmethylcellulose and 1.5 % w/v sodium carboxymethylcellulose exhibited satisfactory swelling, an optimum residence time and promising drug release thus proved to be potential candidate for the development of buccal films for therapeutic use.

In Vitro Characterization, Pharmacokinetics and Reversal of Supratherapeutic Doses of Dabigatran-Induced Bleeding in Rats by a Specific Antibody Fragment Antidote to Dabigatran

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3418-3418 ◽  
Author(s):  
Joanne van Ryn ◽  
Tobias Litzenburger ◽  
Guanfa Gan ◽  
Kelly Coble ◽  
Johanna Schurer
Keyword(s):  
Steady State ◽  
Blood Loss ◽  
Plasma Levels ◽  
Ex Vivo ◽  
Dabigatran Etexilate ◽  
Oral Anticoagulants ◽  
Antibody Fragment ◽  
Terminal Phase ◽  
Thrombin Time

Abstract Abstract 3418 Background: The new oral anticoagulants have demonstrated efficacy and safety in preventing stroke in patients with atrial fibrillation; however, one feature they all share is the lack of a specific antidote in cases of emergency, such as excess bleeding or emergency surgical interventions. A fully humanized monoclonal antibody fragment (Fab) against dabigatran is currently in development for use in the clinic. Methods: The Fab was characterised for binding to dabigatran using Kinexa technology. The dissociation constant (KD) and on-rate (kon) were determined experimentally; the off-rate (koff) was calculated. Inhibition of glucuronidated dabigatran metabolites was tested using a diluted thrombin time assay. Any prothrombotic activity of the Fab was determined by testing the ability to induce coagulation in various assays in human plasma including endogenous thrombin potential (ETP, 5 pM tissue factor) and ecarin chromogenic assay (ECA). Animal studies were approved by local ethics committee and followed principles of laboratory animal care. Pharmacokinetics (PK) of Fab with dabigatran was determined by measuring functional dabigatran, total dabigatran and total Fab in the rat. Dabigatran etexilate (DE) was given to rats every 8 hrs to achieve supratherapeutic levels of dabigatran and induce bleeding. Increasing Fab doses were given to test for bleeding reversal in this rat tail bleeding model. Associated ex vivo clotting and plasma levels of dabigatran and Fab were measured. Results: The Fab has a very tight binding affinity to dabigatran, with a Kd of 2 pM, ∼350-fold more potent than the 0.7 nM Kd of dabigatran binding to thrombin. This binding has a rapid kon (1.5×106 M−1s−1) and a slow koff (3×10−6s−1), resulting in a calculated complex half life (t½) of ∼64 hrs. IC50 of Fab inhibition of 7nM dabigatran was 3.5 nM. Acylglucuronidated dabigatran (7nM) was inhibited by an IC50 of 2nM Fab. There was no effect on thrombin generation when concentrations up to 3 mg/mL Fab were added to plasma (ETP, 0.89-fold of control), in contrast an activated prothrombinase complex concentrate, FEIBA, resulted in 2.06 increase of ETP with 0.8 U/mL. ECA was also not elevated vs control. The PK of the Fab in rats showed a short initial phase t1/2 (0.24 h) and a longer terminal phase t1/2 (5.8 h). Clearance was low (1.95 mL/min/kg), and the steady-state volume of distribution small (0.0688 L/kg; only slightly larger than plasma volume). The PK of the Fab was not affected by dabigatran. DE (30 mg/kg p.o.) given in 8 hr intervals achieved steady state levels in the rat, with dabigatran plasma levels of 1500 nM (∼750 ng/mL), and resulted in an ∼2-fold prolongation of tail cut bleeding time. There was a rapid, dose-dependent decrease in blood loss after i.v. injection of Fab, which was maintained for 6 hrs after the highest dose of Fab. Anticoagulation (clotting ex vivo) was also reversed. Treating rats with warfarin (0.5 mg/kg p.o.) over 3 days resulted in a 2-fold elevation of blood loss. As expected, similar doses of Fab had no effect on reversing this elevated blood loss. Conclusions: These data show the specificity and selectivity of the antibody fragment for dabigatran. Administration of the Fab after DE resulted in a safe and rapid reversal of blood loss, which correlated with reversal of ex vivo clotting tests. Thus this agent holds promise for reversing dabigatran-induced anticoagulation and bleeding effects. It is currently under development for clinical use. Disclosures: van Ryn: Boehringer Ingelheim: Employment. Litzenburger:Boehringer Ingelheim: Employment. Gan:Boehringer Ingelheim: Employment. Coble:Boehringer Ingelheim: Employment. Schurer:Boehringer Ingelheim: Employment.

SSR182289A, a Novel, Orally Active Thrombin Inhibitor: In Vitro Profile and ex Vivo Anticoagulant Activity

2002 ◽  
Vol 303 (3) ◽  
pp. 1189-1198 ◽  
Author(s):  
Christopher N. Berry ◽  
Gilbert Lassalle ◽  
Catherine Lunven ◽  
Jean-Michel Altenburger ◽  
Frédérique Guilbert ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Thrombin Inhibitor ◽  
Orally Active

Slight differences in sulfation of algal galactans account for differences in their anticoagulant and venous antithrombotic activities

2008 ◽  
Vol 99 (03) ◽  
pp. 539-545 ◽  
Author(s):  
Roberto Fonseca ◽  
Stephan-Nicollas Oliveira ◽  
Fábio Melo ◽  
Maria Pereira ◽  
Norma Benevides ◽  
...  
Keyword(s):  
Body Weight ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Experimental Models ◽  
Coagulation System ◽  
Activate Factor ◽  
Factor Xii ◽  
Low Doses ◽  
High Doses ◽  
Recalcification Time

SummaryWe compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis.These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns.As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities.SG from G.crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats.These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.

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