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.

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.

scholarly journals Anticoagulant Properties of a Green Algal Rhamnan-type Sulfated Polysaccharide and Its Low-molecular-weight Fragments Prepared by Mild Acid Degradation

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 445 ◽  
Author(s):  
Xue Liu ◽  
Peng Du ◽  
Xiao Liu ◽  
Sujian Cao ◽  
Ling Qin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharide ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Acid Degradation ◽  
Molecular Weights ◽  
Green Algal

The active sulfated polysaccharide from seaweed possesses important pharmaceutical and biomedical potential. In the study, Monostroma sulfated polysaccharide (MSP) was obtained from Monostroma angicava, and the low-molecular-weight fragments of MSP (MSP-Fs: MSP-F1–MSP-F6) were prepared by controlled acid degradation. The molecular weights of MSP and MSP-F1–MSP-F6 were 335 kDa, 240 kDa, 90 kDa, 40 kDa, 24 kDa, 12 kDa, and 6.8 kDa, respectively. The polysaccharides were sulfated rhamnans that consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ units with partial sulfation at C-2 of →3)-α-l-Rhap-(1→ and C-3 of →2)-α-l-Rhap-(1→. Anticoagulant properties in vitro of MSP and MSP-F1–MSP-F6 were evaluated by studying the activated partial thromboplastin time, thrombin time, and prothrombin time. Anticoagulant activities in vivo of MSP and MSP-F4 were further evaluated; their fibrin(ogen)olytic activities in vivo and thrombolytic properties in vitro were also assessed by D-dimer, fibrin degradation products, plasminogen activator inhibitior-1, and clot lytic rate assays. The results showed that MSP and MSP-F1–MSP-F4 with molecular weights of 24–240 kDa had strong anticoagulant activities. A decrease in the molecular weight of MSP-Fs was accompanied by a decrease in the anticoagulant activity, and higher anticoagulant activity requires a molecular weight of over 12 kDa. MSP and MSP-F4 possessed strong anticoagulant activities in vivo, as well as high fibrin(ogen)olytic and thrombolytic activities. MSP and MSP-F4 have potential as drug or helpful food supplements for human health.

In Vitro Characterization Of Low Molecular Weight Fractions Of Heparin

1981 ◽  
Author(s):  
Jawed Fareed ◽  
Harry L Messmore ◽  
Daniel A Walz ◽  
Jean Choay ◽  
J C Lormeau
Keyword(s):  
Molecular Weight ◽  
The Other ◽  
Low Molecular Weight ◽  
In Vitro Test ◽  
Thrombin Time ◽  
Specific Test ◽  
Platelet Factor ◽  
At Iii ◽  
Heparin Fractions

Numerous extraction, chromatographic (ion exchange, gel, and affinity), chemical and enzymatic degradation methods have been employed to obtain heparin fractions. The present assays to evaluate potency (e.g. pharmacopeial and coagulant) do not truly reflect the antithrombotic properties of these fractions. In addition, the synthetic peptide substrate based assays to measure the anti Xa activity do not correlate with the coagulant anti Xa assays. We have developed an in vitro test battery to evaluate low molecular weight heparin fractions. Porcine mucosal heparin fractions are assayed for anti Xa activity in coagulant and amidolytic assays and the results are expressed as a ratio. The effect of these fractions on coagulant assays such as prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), Stypven time (ST) on freshly prepared normal human plasma (NHP) is determined The retention characteristics of these fractions on platelet factor 4 and AT-III bound sepharose columns were also determined. We have compared the extracted and chemically depolymerized heparin fractions and found that the anti Xa activity doesn’t always correlate with the other parameters studied. The extracted fractions were slightly stronger in the USP assays and showed a biphasic retention on the PF-4 column whereas the chemically depolymerized product showed only one peak. On the other hand, on the AT-III column both fractions showed similar elution patterns. Our studies suggest that heparin and its fractions exhibit differential behavior on various assays and a specific test may not be used as an index of the potency of their antithrombotic effects. Furthermore, the potency of these fractions should be stated on a weight basis when evaluated in the in vivo animal models rather than in terms of a specific test (e.g. anti Xa activity and US Pharmacopeial assays).

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

Defibrotide Augments the Anticoagulant Actions of Heparin and Low Molecular Weight Heparins

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4086-4086
Author(s):  
Jawed Fareed ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Cafer Adiguzel ◽  
Massimo Iacobelli ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Synergistic Effects ◽  
Low Molecular Weight ◽  
Inhibitory Effects ◽  
Clotting Time ◽  
Low Molecular Weight Heparins ◽  
Significant Prolongation ◽  
Antithrombotic Effects

Abstract Defibrotide represents a polydeoxyribonucleotide derived antithrombotic and antiischemic drug, which has been used in the management of vascular disorders and is currently being developed in other clinical indications. Defibrotide is a polyelectrolyte-based agent with target effects on endothelium, platelets, and blood cells. In addition, the aptameric consensus sequences in the nucleotides exhibit inhibitory effects towards thrombin and related proteases. In the anticoagulant assays defibrotide exhibits relatively weak effects (&lt;5 USP U/mg). These studies were undertaken to study whether there is an interaction between defibrotide and unfractionated heparin (UFH) in various systems of anticoagulation. The interaction of defibrotide with commercially available low molecular weight heparins (LMWHs), enoxaparin and dalteparin, was also studied. For the first investigation, to evaluate the effect of defibrotide on the anticoagulant effects of UFH, native whole blood freshly drawn from human volunteers (n = 20) was supplemented with UFH at a fixed concentration of 5 μg/mL (0.8 U/mL), and graded amounts of defibrotide were added in a concentration range of 12.5 – 100 μg/mL. The whole blood celite Activated Clotting Time test (ACT) and the thrombin generation markers fibrinopeptide A (FPA), thrombin-antithrombin complex (TAT), and prothrombin fragment 1.2 (F1.2) were measured. Parallel controls with saline were included. While defibrotide did not produce a significant prolongation of the ACT compared to saline (128 ± 9 s vs 132 ± 7 s), it produced a concentration-dependent increase in the heparinized whole blood leading to an almost doubling of the anticoagulant action of UFH (248 ± 19 s vs 418 ± 21 s). Additional studies carried out by varying the concentrations of the two agents also revealed supraadditive to synergistic effects. Defibrotide also augmented the inhibitory effects of UFH on thrombin generation markers in a concentration-dependent fashion. Similar studies carried out with the two LMWHs did not reveal a similar interaction in the anticoagulant assays such as the ACT; however, significant interactions between defibrotide and the LMWHs were observed in the thrombin generation studies. For the second investigation, studies were carried out using plasma samples collected from heparinized patients (aPTT of 50 – 100 s). These studies also revealed that supplementation of defibrotide augmented the anticoagulant effects of UFH in a concentration-dependent fashion. While defibrotide at 12.5 μg/mL did not significantly increase the aPTT of normal plasma, when supplemented to heparinized plasmas (n = 50 with aPTT of 64.6 ± 14.0 s) it produced a strong prolongation of the clotting time (96.1 ± 20.6 s). In the third investigation, animal models of thrombosis including the rat jugular vein clamping model, demonstrated an augmentation of the antithrombotic effects of intravenously administered UFH by defibrotide. However, no augmentation of the hemorrhagic effect was observed in the rat tail bleeding model. These studies demonstrate that defibrotide exhibits a strong anticoagulant interaction with UFH and to a lesser degree LMWH. While the combination of defibrotide and UFH exhibits enhanced anticoagulant/antithrombotic activities, it does not exhibit any alteration of the hemorrhagic profile. These studies clearly suggest that defibrotide can be combined with UFH to achieve a superior anticoagulant approach with better safety/efficacy profile.

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.

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