Faculty Opinions recommendation of Homogeneous low-molecular-weight heparins with reversible anticoagulant activity.

2015 ◽  
Author(s):  
Yukishige Ito
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins

Synthesis and Anticoagulant Activity of Low-Molecular-Weight Heparins

2004 ◽  
Vol 38 (2) ◽  
pp. 82-85
Author(s):  
P. P. Stolbushkina ◽  
G. E. Bannikova ◽  
N. N. Drozd ◽  
V. A. Makarov ◽  
V. P. Varlamov ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins

Structural features of low-molecular-weight heparins affecting their affinity to antithrombin

2009 ◽  
Vol 102 (11) ◽  
pp. 865-873 ◽  
Author(s):  
Antonella Bisio ◽  
Davide Vecchietti ◽  
Laura Citterio ◽  
Marco Guerrini ◽  
Rahul Raman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Nmr Spectra ◽  
Biological Activities ◽  
Anticoagulant Activity ◽  
2D Nmr ◽  
Structural Features ◽  
Size Exclusion ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
2D Nmr Spectra

SummaryAs part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin,Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT).The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions,molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*.Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.

The Additive Effect of Low Molecular Weight Heparins on Thrombin Inhibition by Dermatan Sulfate

1993 ◽  
Vol 70 (03) ◽  
pp. 443-447 ◽  
Author(s):  
Benilde Cosmi ◽  
Giancarlo Agnelli ◽  
Edward Young ◽  
Jack Hirsh ◽  
Jeffrey Weitz
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Additive Effect ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Sds Page

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

Comparative Studies on An Anti-Xa Enriched Ultra Low Molecular Weight Heparin with Bemiparin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4394-4394
Author(s):  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Josephine Cunanan ◽  
Walter Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Anticoagulant Activity ◽  
The Other ◽  
Positive Interactions ◽  
Low Molecular Weight ◽  
Significant Activity ◽  
Thrombin Time ◽  
Low Molecular Weight Heparins

Abstract Abstract 4394 Most low molecular weight heparins (LMWHs) have a mean molecular weight in the range of 4–6 kDa and anti-Xa/IIa ratios of 3–6. Further depolymerization of porcine mucosal heparin results in the generation of Ultra low molecular weight heparins (ULMWHs) with a molecular weight range of 2–4 kDa with proportionately decreased anti-Xa and anti-IIa activities. Bemiparin (Rovi, Madrid, Spain) represents one such ULMWH. AVE 5026 (Sanofi-Aventis, Paris, France) is a unique ULMWH (2.5 kDa) which exhibits higher affinity to antithrombin (AT) and therefore, enhanced anti-Xa activity. Because of the compositional differences between these two agents, it was hypothesized that each of these agents will have distinct anticoagulant, antiprotease and thrombin generation effects. Each of these agents was supplemented to native whole blood. Anticoagulant activity was measured using ACT, TEG, PT, APTT, thrombin time and Heptest assays. Similar studies were carried out in plasma. Amidolytic assays were used to determine the anti-Xa and anti-IIa activities. Both agents were also tested for the interactions with heparin cofactor II (HC II) and AT and were compared in the HIT antibody screening assay using platelet aggregation. In whole blood clotting assays bemiparin showed a strong anticoagulant activity in comparison to AVE 5026. Both agents also exhibited assay dependent differences in the APTT, heptest and thrombin time assays. AVE 5026 exhibited a higher anticoagulant activity in the heptest whereas bemiparin showed a stronger anticoagulant effect in the other clot based assays. In the amidolytic anti-Xa assay, AVE 5026 showed an activity of 156U/mg compared to 86 U/mg for bemiparin. In the anti-IIa assay bemiparin showed a higher activity (10 U/mg) in comparison to AVE 5026 (3.2 U/mg). The calculated Xa/IIa ratio of AVE 5026 was > 48, whereas it was 8.6 for bemiparin. While bemiparin exhibited interactions with HC II, AVE 5026 did not show significant activity in the tested concentrations (anti-IIa – IC50: 1.10±.45 μ M and >3.44±.00 μ M, respectively). On the other hand, AVE 5026 exhibited stronger interactions with AT in comparison to bemiparin (anti-FXA – IC50: .223±.03 μ M and .894±.06 μ M, respectively). Interestingly, heparinase digestion of the two products resulted in a complete loss of anti-IIa activity, but residual anti-Xa activity was found. AVE 5026 exhibited stronger anti-Xa interactions even after heparinase digestion. In the heparin induced platelet aggregation assay at 2.5 μ g/ml, bemiparin showed a relatively higher prevalence of positive interactions with HIT antibodies, whereas AVE 5026 showed a much lower prevalence (slope; AVE 5026 compared bemiparin, p=0.012). Bemiparin exhibited greater platelet factor 4 neutralization in comparison to AVE 5026. These studies clearly demonstrate that while bemiparin behaves like a typical ULMWH, AVE 5026 behaves differently in the different assays. Moreover, the oligosaccharide composition of the two products, in terms of distribution profile structure, is also different. Therefore, AVE 5026 does not represent a typical depolymerized ULMWH and is expected to exhibit a distinct pharmacologic and clinical profile. Disclosures: Hoppensteadt: Sanofi-Aventis: Research Funding.

scholarly journals Standard and Method Independent Units for Heparin Anticoagulant Activities

1993 ◽  
Vol 70 (05) ◽  
pp. 724-728 ◽  
Author(s):  
H C Hemker ◽  
S Béguin
Keyword(s):  
Molecular Weight ◽  
Catalytic Effect ◽  
Anticoagulant Activity ◽  
Quantitative Description ◽  
Factor Xa ◽  
Assay Method ◽  
Low Molecular Weight ◽  
Laboratory Practice ◽  
Low Molecular Weight Heparins ◽  
At Iii

SummaryIt is discussed why the current USP unit of heparin anticoagulant activity necessarily will render inaccurately the anticoagulant activities of low molecular weight heparins. It is shown that the outcome is bound to vary with the method used for comparison of the sample and the standard and with the nature of the standard used. As an alternative we define a unit of heparin in terms of anti-factor Xa- and antithrombin-activity that is independent of the heparin standard and of the assay method, but that is based upon a quantitative description of the catalytic effect of heparin on AT III mediated thrombin- and factor Xa breakdown. Expression of the results of existing anti-factor Xa- and antithrombin tests in terms of these units will allow to express heparin levels in plasma in terms of concentrations of active anticoagulant material. This approach makes it possible to separate heparin pharmacodynamics from heparin pharmacokinetics. Introduction of this unit does not require adaptation of current laboratory practice but changes the way in which the results obtained are expressed.

Generic and Branded Low Molecular Weight Heparins Can Be Differentiated Based on Biologic and Pharmacologic Assays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1885-1885
Author(s):  
Walter P. Jeske ◽  
Paul Ackerman ◽  
Amanda Drenth ◽  
Jeanine M. Walenga ◽  
Mamdouh Bakhos
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Complex Nature ◽  
Low Molecular Weight ◽  
Functional Assay ◽  
Low Molecular Weight Heparins ◽  
Biologic Drugs ◽  
Branded Drug

Abstract Several generic versions of the low molecular weight heparins enoxaparin and dalteparin have become available in different parts of the world and several products are currently under review by the FDA. At this time, there are no regulatory guidelines for defining equivalence of complex biologic drugs. Because of their complexity and hybrid nature (biologic and chemical), the available generic versions may not be acceptable by using currently applicable generic criteria. Due to their complex nature, the pharmacologic effect of these may not be similar to the branded product. Therefore, additional methods are needed to equate these drugs with the branded products. In this study, multiple lots of Lovenox or generic enoxaparin such as Lupenox (Lupin Pharma, India), Dripanina (Ariston, Brazil), Dilutol (Lazar, Argentina), Clenox (Pharmayect, Columbia), and Cutenox (Gland Pharma, India) and generic versions of dalteparin such as Daltehep (Gland Pharma, India) were supplemented to human plasma in the absence or presence of protamine sulfate (PS). Anticoagulant activity was measured using the aPTT, Heptest, PiCT assays. Antithrombin and anti-factor Xa activity were assessed by amidolytic assays. Effects on thrombin generation and the activation of thrombin activatable fibrinolytic inhibitor (TAFI) were determined by functional assay. At prophylactic levels of LMWH, no differences in anticoagulant or antiprotease activities were observed between generic and branded LMWHs. At therapeutic concentrations, significantly higher anticoagulant and antiprotease activities were observed with Lovenox. Assay-dependent variations in PS neutralization of antiprotease and anticoagulant activities were observed at higher concentrations of the generic versions of enoxaparin and dalteparin. Significant differences were noted in the effects of these agents on thrombin generation and the activation of TAFI. Current generic LMWHs possess some differences from branded LMWHs in known biologic properties of heparin. Variations in PS neutralization raise the question as to whether the interaction with other plasmatic proteins also differs from that of the branded drug. These results emphasize a need to consider multiple functional parameters when defining bioequivalence of complex biologic drugs and underscore the importance of further pharmacologic studies involving animal models and human clinical trials. Such approaches can be incorporated in the recommendations to develop guidelines for the approval of the generic versions of low molecular weight heparins.

scholarly journals Homogeneous low-molecular-weight heparins with reversible anticoagulant activity

2014 ◽  
Vol 10 (4) ◽  
pp. 248-250 ◽  
Author(s):  
Yongmei Xu ◽  
Chao Cai ◽  
Kasemsiri Chandarajoti ◽  
Po-Hung Hsieh ◽  
Lingyun Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4324-4324
Author(s):  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Jeanine M. Walenga ◽  
Josephine Cunanan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Confidence Interval ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Serotonin Release ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Safety And Efficacy ◽  
Response Data

Abstract Abstract 4324 Introduction: Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods: Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results: A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions: These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures: Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

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