Comparison of Branded Enoxaparin (Lovenox) and a Biosimilar Version of Enoxaparin (Fibrinox)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4385-4385
Author(s):  
Walter Jeske ◽  
Elizabeth McGeehan ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Average Molecular Weight ◽  
Molecular Profile ◽  
Distribution Analysis ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Branded Product ◽  
Inhibition Of Thrombin

Abstract Abstract 4385 Several biosimilar versions of branded enoxaparin (Lovenox, Sanofi-Aventis, Paris, France) have recently become available throughout the world. These biosimilar enoxaparin preparations are distributed by multiple suppliers in Asia and in North and South America. Enoxaparin represents a complex mixture of oligosaccharides obtained by alkaline depolymerization of porcine mucosal heparin. It is the most widely used low molecular weight heparin which has been validated for clinical use in multiple indications. While the molecular profile and anti-Xa potencies of some of the biosimilar versions of enoxaparin are comparable, product based differences have been reported amongst some of the biosimilar versions of enoxaparin. The purpose of this study was to compare the biochemical and pharmacologic profile of one biosimilar version of enoxaparin, namely Fibrinox (Sandoz SA, Buenos Aires, Argentina) with the branded product Lovenox. The products were compared in equigravimetric amounts, assuming equivalent potency (100 AXa U/mg). Both products exhibited comparable molecular weight profiles in terms of average molecular weight and oligosachharide distribution. Analysis of the antithrombin binding hexasaccharide fractions of Fibrinox and Lovenox indicated the presence of eight distinct hexasaccharides. The relative proportions these hexasaccharides differed between Fibrinox and Lovenox. The anti-Xa and anti-IIa activities were comparable. In the whole blood clot-based assays such as TEG and ACT, both agents produced similar anticoagulant effects. In the plasma based assays such as the APTT, Heptest and thrombin time, both products showed comparable anticoagulant effects in the normal human pooled plasma samples. However, in plasma samples collected from patients with liver disease who were apparently anticoagulant free, the two products showed differences in their anticoagulant effects in the APTT assay (p<0.05). In the TF mediated thrombin generation assay, Fibrinox produced a stronger inhibition of thrombin generation compared to Lovenox (IC50; Fibrinox, 1.6 μ g/ml, Lovenox 2.2 μ g/ml). No differences were observed between the two products in the agonist induced platelet aggregation assays. However in the 14C serotonin release study, Fibrinox produced a stronger HIT serum mediated 14C release (p<0.05). Differences in the fibrinokinetic profile and the inhibition of thrombin activatable fibrinolytic inhibitor activation were observed with these LMWHs. These studies suggest while both the molecular profile and the pharmacopoeial potency of Fibrinox is similar to the branded product, these drugs can be differentiated in some of the other assays and should be evaluated in terms of additional pharmacologic mechanisims to demonstrate bioequivalence. Disclosures: No relevant conflicts of interest to declare.

Differential inhibition of thrombin generation by vitamin K antagonists alone and associated with low-molecular-weight heparin

2009 ◽  
Vol 102 (07) ◽  
pp. 42-48 ◽  
Author(s):  
Grigoris T. Gerotziafas ◽  
Charlotte Dupont ◽  
Alex C. Spyropoulos ◽  
Mohamed Hatmi ◽  
Meyer M. Samama ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Vitamin K ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Vitamin K Antagonists ◽  
Low Molecular Weight ◽  
Plasma Samples ◽  
Thrombin Generation Assay ◽  
International Normalised Ratio ◽  
Inhibition Of Thrombin

SummaryVitamin K antagonists (VKA) treatment starts with co-administration of low-molecular-weight heparin (LMWH). The anticoagulation induced by the two drugs is still not well determined. In the present study we used thrombin generation assay to evaluate the hypo-coagulation induced by treatment with VKA and by the combination of VKA with LMWH. Tissue factor triggered thrombin generation in platelet-poor plasma was assessed in samples from 15 healthy volunteers, 97 samples from patients treated with VKA and 41 samples from patients receiving enoxaparin and VKA. Patients were classified according to international normalised ratio (INR) level (<2, 2–3 and >3).In plasma samples from patients treated with VKA having INR 2–3 the inhibition of thrombin generation reached 50% compared to controls. In samples with INR>3 this inhibition was 80%. In samples from patients receiving both LMWH and VKA, thrombin generation was significantly decreased compared to the controls and VKA group. In samples with an INR 2–3 obtained from patients treated with LMWH and VKA, the inhibition of thrombin generation was similar to that observed in samples with an INR>3 obtained from VKA treated patients. Thrombin generation assay is sensitive to detect the global the anticoagulant effect produced by the association of LMWH and VKA. For equal INR dual anticoagulant treatment induces significantly more profound inhibition of thrombin generation compared to treatment with VKA alone. The clinical relevance of this observation merits to be studied in prospective studies in patients with defined indications of anticoagulant therapy.

Persistant Inhibition of Thrombin Generation After Intravenous Administration of Enoxaparin in Primates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2095-2095
Author(s):  
Evangelos Litinas ◽  
Angel Gray ◽  
Nasir Sadeghi ◽  
Josephine Cunanan ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Clinical Effects ◽  
Plasma Samples ◽  
Thrombin Inhibition ◽  
Biologic Effects ◽  
Antithrombotic Effects ◽  
Sustained Inhibition ◽  
Inhibition Of Thrombin

Abstract Abstract 2095 Poster Board II-72 The biologic half life (T12) of low molecular weight heparin (LMWH) is usually measured in terms of the circulating anti-Xa levels. Enoxaparin represents an unique LMWH whose biologic T12 is relatively longer than most LMWHs. Moreover, it is known that the antithrombotic effects of this agent last longer in comparison to the measurable circulating anti-Xa levels. Therefore besides the anti-Xa activity, additional non-measurable biologic effects are contributory to the clinical effects of this agent. Plasma based thrombin generation assays have recently become available to assess the effects of LMWHs such as enoxaparin. In these assays blood plasma samples are activated using different activators and the generated thrombin inhibition is measured. To measure the time course of thrombin generation inhibitory activity after an IV bolus dose of 0.5 mg/kg of enoxaparin into groups of primates (n=6-8), a commercially available thrombin generation method was employed (Technoclone, Vienna, Austria/DiaPharma, West Chester,OH). Blood samples were drawn from each of the primates injected at varying time points for up to 28 hours. A thromboplastin/phospholipids based reagent was used to generate thrombin and the results were recorded in terms of nm of thrombin formed. The baseline values ranged from 500-900 nm (710±60 nm), although a complete inhibition of thrombin generation was noted at 1 hour (24±8 nm), a slow and gradual reduction in the thrombin generation inhibition was noted with a T12 of 9 hours. Even at 28 hours after the administration of enoxaparin, sustained inhibition of thrombin generation was noted (30-50%). Interestingly, the circulating anti-Xa and anti-IIa activity gradually diminished to an almost non-detectable level at 6 hours. These studies suggest that enoxaparin produces antithrombotic actions by multiple mechanisms. Furthermore thrombin generation methods in plasma samples may provide a more sensitive assay for the monitoring of the effect of LMWH. Disclosures: No relevant conflicts of interest to declare.

Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1251-1251
Author(s):  
Debra Hoppensteadt ◽  
Walter Jeske ◽  
Angel Gray ◽  
Jeanine M. Walenga ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Molecular Profile ◽  
Thrombin Time ◽  
Time Period ◽  
Pharmacodynamic Profile ◽  
The Us ◽  
Fibrinolysis Inhibitor ◽  
Tissue Factor Pathway

Abstract Abstract 1251 Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Generic Version of Recombinant FVIIa Is Similar to the Branded Product (NovoSeven)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4746-4746
Author(s):  
Nasir Sadeghi ◽  
Paul O'Malley ◽  
Daniel Kahn ◽  
Debra Hoppensteadt ◽  
Jawed Fareed
Keyword(s):  
Protein Content ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Generic Version ◽  
Factor Vii ◽  
Molecular Profile ◽  
The Us ◽  
Branded Product

Abstract Background: Commercially available recombinant factor VIIa (Novoseven) is widely used in the management of hemophilia patients with inhibitors. Recently several generic versions of recombinant VIIa (rFVIIa) have become available. The generic versions of rFVIIa are claimed to be biosimilar to the barnded Novoseven (Novo Nordisk, Copenhagen, Denmark). The purpose of this study is to compare the US and European Novoseven products with a generic version of rFVIIa namely, Aryoseven (Aryogen, Tehran, Iran). Methods: Four commercially available random lots of Novoseven were obtained from the US and European sources. Four different batches of Aryoseven were obtained from Aryogen. All individual rFVIIa preparations were diluted to obtain working concentrations of 100, 10, 1 and 0.1 ug/ml. Protein content (Lowry's method), molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), factor VII related antigen level (FVII:Ag), factor VII correction studies in depleted plasma and thrombin generation (TG) studies were carried out. In addition, VIIa/tissue factor mediated thrombin generation studies were carried out in various prothrombin complex concentrates such as Beriplex and Prothromplex. Results: The protein content and SELDI mass spectrophotometric profile of all 4 rFVIIa preparations were comparable. There was no differences in the Novoseven obtained from the US and European sources. The GEP of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 25 KDa. The FVII related antigen levels were also comparable in the Novoseven and Aryoseven preparations. Supplementation of both the Novoseven and Aryoseven preparations at 10 and 100 ug/ml resulted in a comparable correction of the factor VII deficient plasma as measured by PT(INR). Thrombin generation was comparable in the branded and generic product. Conclusions: These results demonstrate that the US and European Aryoseven are comparable. Four batches of Novoseven and 4 individual clinical batches of Aryoseven were found to be comparable. When the US purchased Novoseven preparation was compared with the European Novoseven product, no differences were noted. Thus, the generic Aryoseven is biosimilar to barnded Novoseven and warrant in vivo validation studies. Disclosures No relevant conflicts of interest to declare.

Resourcing of Heparin and Low Molecular Weight Heparins from Bovine, Ovine, and Porcine Origin. Studies to Demonstrate the Biosimilarities

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4733-4733 ◽  
Author(s):  
Debra Hoppensteadt ◽  
Paula Maia ◽  
Alice Silva ◽  
Emmanuel Kumar ◽  
Nil Guler ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Size Exclusion ◽  
Molecular Profile ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Low Molecular Weight Heparins ◽  
Functional Assays ◽  
Induced Aggregation ◽  
Porcine Origin

Abstract Introduction: The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. At the same time the demand for heparins has increased requiring alternate sources to obtain heparin and LMWH. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The pharmaceutical industry has already initiated programs to manufacture Bovine, Ovine, and Porcine heparins and depolymerized enoxaparin from these products some of which are currently in various phases of development. The purpose of this study is to compare 5 individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins. Methods: The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion method. A narrow range calibration method was used for comparing the molecular weight of heparin, whereas the EP method was used to cross-reference the molecular weight of enoxaparins. The anticoagulant potency was measured use clot based methods such as aPTT and Thrombin Time. Chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The interaction between AT and heparins and enoxaparin were investigated in a purified biochemical system, using AT supplemented buffered assay. Thrombin Generation inhibition studies were carried out using a flourometric method (Technoclone, Vienna, Austria). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry. Results: The molecular profile of the Bovine, ovine, and porcine heparins and enoxaparin were almost identical. In the clot based assays, such as PT and PTT, Porcine and Ovine heparin produce consistently comparable anticoagulant effects, which were stronger in comparison to the bovine derived heparins. In contrast, the enoxaparins derived from these three sources showed minimal differences. In the amidolytic anti Xa and IIa assays both ovine and porcine heparins produced similar inhibitory effects, whereas the bovine heparin exhibited lower activity. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of the Porcine and Ovine heparins ranged from 180 to 190u/mg, whereas the Bovine was found to be 130-140 u/mg. The anti-Xa - IIa ratio for the heparin were comparable. The ovine and porcine enoxaparin exhibited comparable potencies which ranged 94-110 u/mg whereas bovine enoxaparin was slightly lower 80-87 u/mg. However the antiXa and anti-IIa ratios were comparable. The AT mediated inhibition of factor Xa and anti-IIa was stronger with heparins in comparison to the enoxaparins. Similarly heparins produced stronger inhibition of thrombin generation in comparison to the enoxaparin. In the HIT screening there was no difference between the HIT responses in the heparins from different species. Similar results were obtained with enoxaparins. Conclusions: These studies show that while bovine, ovine and porcine heparins and enoxaparins exhibit comparable molecular profiles however in some of the functional assays bovine heparin and enoxaparin exhibited somewhat lesser potencies especially in the pharmacopeial assays. No differences were noted in the HIT antibody interactions among heparins and enoxaparins from different species. These studies demonstrate that ovine and porcine heparins are biosimilar and can be developed as such for clinical purposes. The bovine derived heparins exhibit slightly weaker potencies in functional assays despite comparable molecular profile. Potency adjustment for in vivo usage may be required to obtain comparable anticoagulant responses for the bovine heparin and enoxaparin. Disclosures No relevant conflicts of interest to declare.

Differential Digestion of Different LMWHs by Heparinase-I and Heparinase-II: Drug Developmental Implications.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4084-4084
Author(s):  
Jyothi Maddineni ◽  
Walter P. Jeske ◽  
Omer Iqbal ◽  
Debra A. Hoppensteadt ◽  
Jawed Fareed
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
High Performance ◽  
Uronic Acid ◽  
Specific Binding ◽  
Average Molecular Weight ◽  
Uv Detection ◽  
Molecular Profile ◽  
Heparinase I ◽  
Differential Digestion

Abstract The purpose of this study was to determine the differential digestion of two different batches of branded enoxaparin (Aventis, USA), three generic versions of enoxaparin (GlandPharma, India; Lazar, Argentina; and Biochemie, Brazil), dalteparin (Pfizer, USA) and tinzaparin (Leo, USA) by heparinase-I and heparinase-II. Heparinase-I (Ibex Tech., Montreal, Canada) and heparinase-II (Ram Sasisekharan, MIT, Cambridge, MA) were isolated from Flavobacterium heparinum. The substrate specificity of these enzymes has been inferred from the reducing and non-reducing terminal structures of the di and oligosaccharides formed by digesting heparin. Heparinase-I cleaves the glucosaminidic linkage in GlcN (N-sulfate) a 1–4 IdceA (2-sulfate) and endures C-6 sulfation of hexosamine unit. More susceptibility of polymers such as heparin than oligomers to this enzymatic depolymerization indicates the size dependency of this enzymatic activity. Heparinase-II cleaves all the glucosaminidic linkages in heparin independent of O-and/or N-sulfation as well as the type of uronic acid residue. The non-sulfated substrates are somewhat resistant to this enzyme. The glucosaminidic linkage adjacent to a 3-O-sulfated GlcN residue and the innermost glucosaminidic linkage right next to the glycosaminoglycan-protein linkage region of proteoglycan are resistant to this enzymatic activity (Sugahara et al., 1994, Glycobiology 4, 535–544). In this study, several low molecular weight heparins (LMWHs) produced from different depolymerization methods of unfractionated heparin were digested with heparinase-I and heparinase-II to determine the differential digestion of these two enzymes. Eight different LMWHs with average molecular weight (MW) ranging from 3425 to 6281 Da (in UV detection at 205nm) were prepared at a concentration of 10mg/ml in 0.3M Na2SO4. Each LMWH was incubated with these enzymes (1.0 U/mL) separately for 30 minutes at 37° C in the presence of calcium Following the incubation, the samples were heated at 100° C to inactivate enzymatic activity. The molecular weight profiling of these samples was determined by using a gel permeation chromatography-high performance liquid chromatography (GPC-HPLC) system with UV detection at 205nm. A narrow range calibration method comprised of oligosaccharides and polysaccharides was used to determine the relative molecular profile of the native and digested products. The LMWHs were digested to LMW oligosaccharides with average MW of 1510± 275 Da with heparinase-I and 3071± 1044 Da with heparinase-II. The extent of digestion observed with all the LMWHs was more with heparinase-I than heparinase-II. This may be due to the different specific binding sites available for these enzymes and the requirement of sulfation at different positions in GlcN/uronic acid residues. All the LMWHs were equally digested into oligosaccharides (di, tetra and hexa) with heparinase-I. However heparinase-II resulted in the formation of only hexa, octa and decasaccharides. These results show that the LMWHs were more susceptible to heparinase-I than heparinase-II. The possible reason for the less susceptibility of these compounds to heparinase-II is likely due to the oligosaccharide composition and degree and pattern of sulfation in GlcN/uronic acid residues of these compounds. The heparinase-I and heparinase-II digestion can therefore be used in the profiling of these agents.

Pharmacodynamic Differences of An Anti-Xa Enriched Low Molecular Weight Heparin, AVE 5026 in Comparison to Enoxaparin and Unfractionated Heparin.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4171-4171
Author(s):  
Debra Hoppensteadt ◽  
Angel Gray ◽  
Evangelos Litinas ◽  
Brigitte Kaiser ◽  
Jawed Fareed
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Time Course ◽  
Fold Increase ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Primate Model

Abstract Abstract 4171 AVE5026 (Sanofi-Aventis, Paris, France) represents an anti-Factor (F) Xa enriched ultra low molecular weight heparin (ULMWH) (Mw=2.4 Kda; anti-FXa activity ∼160 U/mg). In comparison to Enoxaparin it has a lower anti-FIIa activity (∼2 U/mg). The oligosaccharide composition of AVE5026 also differs from Enoxaparin and other LMWHs. Besides the molecular and compositional differences, the biologic half-life of AVE5026 (18-20 hours) is significantly longer than Enoxaparin (4-6 hours). In order to compare the other pharmacodynamic differences between AVE5026, Enoxaparin and unfractionated heparin (UFH), a primate model (macaca mulatto) was used since its tissue factor pathway inhibitor (TFPI) profile is comparable to the human response. Individual groups of primates (n=6) were administered with 1 mg/kg SC of either AVE5026, Enoxaparin or UFH. Heptest and APTT measurements were determined on whole blood (WB) and plasma was analyzed for APTT, Heptest, thrombin time (TT), anti-FXa and anti-FIIa effects at varying periods up to 28 hours. TFPI antigen was measured using the assay from Stago (Parsipanny, NJ). Functional TFPI measurements were determined using the kit from American Diagnostica (Stamford, CT). In contrast to UFH, in the WB assays, neither the AVE5026 nor the Enoxaparin produced a strong effect on the APTT and TT, however both demonstrated a strong effect on the heptest assay. AVE5026 produced a much stronger effect with a longer half-life (T½=11 hrs) in comparison to Enoxaparin (T½=6 hrs). In the plasma based systems only UFH produced a measurable effect on the APTT and TT. However, in the heptest and anti-FXa assays, both AVE5026 and Enoxaparin produced a stronger effect, which was much longer with AVE5026 (2-3 fold increase). The plasma time course of TFPI antigen release was longer with AVE5026 in comparison to Enoxaparin and UFH. The ratios of immunologic to functional TFPI levels were also higher in the primates administered with AVE 5026. In the thrombin generation test, AVE5026 produced a sustained effect which lasted longer than Enoxaparin (T½ =16.8 hrs vs. 9.2 hrs.). These results show that AVE5026 produces stronger anti-FXa effects in primates which are associated with a higher circulating level of TFPI and more pronounced suppression of thrombin generation compared to Enoxaparin and UFH. Disclosures: Hoppensteadt: Sanofi-Aventis: Research Funding.

Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5129-5129
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Jeanine M. Walenga ◽  
Bruce E Lewis
Keyword(s):  
Protein Interactions ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Effect ◽  
Thrombin Time ◽  
Clotting Time ◽  
Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

Relieving Inhibition of Prothrombinase By TFPIα: a Procoagulant Activity of Unfractionated, Low Molecular Weight, and Nonanticoagulant Heparins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1478-1478
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Rodney M. Camire ◽  
Umesh R Desai ◽  
Alan E. Mast
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Research Funding ◽  
Thrombin Generation ◽  
Size Dependence ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Direct Inhibition ◽  
Acidic Region ◽  
Inhibition Of Thrombin

Abstract Introduction: Prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa), is inhibited by tissue factor pathway inhibitor (TFPI)α during the initiation of coagulation (Wood JP et al, PNAS 2013). Efficient inhibition of thrombin generation by prothrombinase requires an interaction between the TFPIα basic C-terminus and an acidic region of the FVa B-domain. This acidic region is present in FXa-activated FVa and FVa released from activated platelets, but is rapidly removed by thrombin. Thus, prothrombinase inhibition only occurs during the initiation phase of thrombin generation. As the exosite interaction is charge-dependent, large negatively charged molecules, including unfractionated heparin (UFH), block it, prevent prothrombinase inhibition, and promote thrombin generation. Studies using the negatively charged molecule polyphosphate have suggested a size requirement for blocking this TFPIα activity (Smith SA et al, Blood2010). A similar size-dependence may exist with heparins and could have clinical implications, as currently-used heparins range from long (unfractionated heparin; UFH) to medium (low molecular weight heparins; LMWHs) to short (the antithrombin-binding pentasaccharide fondaparinux). Studies were performed to assess the ability of the LMWHs enoxaparin and dalteparin, fondaparinux, and the nonanticoagulant heparin 2-O, 3-O desulfated heparin (ODSH) to block TFPIα and promote thrombin generation through this mechanism. Methods: TFPIα inhibition of thrombin generation by prothrombinase, assembled with a form of FVa containing the acidic region of the B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, fondaparinux, and ODSH. The effect of these compounds on the direct inhibition of FXa by TFPIα was measured using a FXa chromogenic substrate. The effect of these compounds on thrombin generation in plasma was measured by calibrated automated thrombography using human plasma immunodepleted of antithrombin III and heparin cofactor II (AT3/HCII-depleted plasma). Results: TFPIα inhibited prothrombinase activity (IC50 = 6.8 nM), and UFH blocked this inhibition (IC50 = 12.5 nM or 14.9 nM at 0.5 or 1 U/mL, respectively). Enoxaparin (0.8 U/mL; IC50 = 30.3 nM) and dalteparin (1 U/mL; IC50 = 29.7 nM) appeared to be more effective at reversing TFPIα inhibition. The reason for this apparent enhanced effect of LMWHs compared to UFH is not clear, as UFH and the LMWHs similarly enhanced the direct inhibition of FXa by TFPIα, and the differential activity was also observed when heparins were normalized to saccharide concentration. The same pattern was observed when measuring thrombin generation in AT3/HCII-depleted plasma, with LMWHs being more procoagulant than UFH. Consistent with TFPIα inhibition being charge-dependent, ODSH promoted thrombin generation similarly to LMWHs in both purified systems and AT3/HCII-depleted plasma. In contrast, clinical doses of fondaparinux had no effect in any assay. In a purified system, ~1000 times the clinical dose of fondaparinux was required to promote thrombin generation. Conclusion: There is a size-dependence for blocking TFPIα inhibition of prothrombinase using heparins, as the pentasaccharide has no effect. However, both LMWHs and UFH are sufficiently long to express this procoagulant activity at therapeutic doses. In addition, the nonanticoagulant heparin ODSH blocks prothrombinase inhibition by TFPIα. This procoagulant activity is likely most clinically relevant under conditions of antithrombin deficiency, which may result from sepsis, liver failure, or administration of L-asparaginase. Under any of these conditions, UFH, LMWHs, and ODSH may have unanticipated procoagulant activity mediated by blocking TFPIα. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast:Novo Nordisk: Research Funding.

scholarly journals Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1158-1158
Author(s):  
Fakiha Siddiqui ◽  
Alfonso J Tafur ◽  
Debra Hoppensteadt ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Research Funding ◽  
Thrombin Generation ◽  
Surrogate Marker ◽  
Factor Xa ◽  
Bleeding Complications ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Inhibitory Effects ◽  
Biologic Effects ◽  
Inhibition Of Thrombin

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

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