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

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.

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Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

Blood ◽

10.1182/blood-2020-142861 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-6

Author(s):

Nausheen Baig ◽

Ahmed Kouta ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

Jeanine Walenga ◽

...

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Area Under The Curve ◽

Anticoagulant Effect ◽

Clotting Time ◽

Kinetic Assay ◽

Pharmaceutical Ingredients ◽

Biologic Effects ◽

Mass Basis

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

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Anticoagulant Kinetics Following Bolus Injection of Heparin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657020 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1248-1260 ◽

Cited By ~ 1

Author(s):

Lyle F Mockros ◽

Samuel D Hirsch ◽

Leon Zuckerman ◽

Joseph A Caprini ◽

William P Robinson ◽

...

Keyword(s):

Whole Blood ◽

Blood Clotting ◽

Anticoagulant Effect ◽

Post Injection ◽

Clotting Time ◽

Sequential Samples ◽

Dose Dependent ◽

Blood Clotting Time

SummaryBolus injections of beef-lung heparin at doses of 50, 100 and 200 u/kg body weight were administered to mongrel dogs. Neutralization of the anticoagulant effect was evaluated using sequential samples withdrawn from the animals (in vivo samples) and aliquots from a 100 ml sample withdrawn from the dog at 30 minutes post-injection (in vitro samples). Tests of the activated partial thromboplastin time (APTT) and prothrombin time (PT) did not indicate the degree of anticoagulation. Tests of the whole blood clotting time (WBCT), celite- activated whole blood clotting time (ACT), and celite-activated thromboelastography (ATEG) indicated pronounced hypocoagulability immediately after the injection, followed by a fairly rapid decay in anticoagulability, and a slight Ziype/coagulability at three to four hours post injection. The results from the in vitro ATEG samples were essentially identical to those on the in vivo samples, whereas the in vitro WBCT and ACT generally indicated higher degrees of anticoagulation. Calculated half-lives of the anticoagulant effect are significantly shorter than previously reported, being 18 to 36 minutes, and slightly dose dependent. The decay of the effects, however, does not appear to follow a single exponential curve, dropping very rapidly immediately post-injection and at a somewhat slower rate 60 or more minutes post-injection.

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Comparative Studies On Branded Enoxaparin and a US Generic Version of Enoxaparin.

Blood ◽

10.1182/blood.v120.21.2264.2264 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2264-2264

Author(s):

Jeanine M. Walenga ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

Josephine Cunanan ◽

Vicki Escalante ◽

...

Keyword(s):

Molecular Weight ◽

Whole Blood ◽

Research Funding ◽

Thrombin Generation ◽

Ex Vivo ◽

Anticoagulant Effect ◽

Individual Subject ◽

The Us

Abstract Abstract 2264 Background: Low molecular weight heparins (LMWHs) are complex biologic drugs whose heterogeneity in saccharide chain length and in the composition (sulfate, acetyl), content, and location of functional groups can impact their multiple biologic activities. Enoxaparin (Lovenox®) is validated for multiple indications and is the most widely used LMWH in the US. Several generic versions of enoxaparin are currently available in the US. We undertook this study to compare the activity profile of branded and a generic enoxaparin. Methods: Five batches each of branded (B; Sanofi-aventis; Bridgewater, NJ) and generic (G; Sandoz US; Princeton, NJ) enoxaparin were studied. Drugs were purchased through hospital pharmacies as pre-filled syringes containing 40 mg drug. The molecular weight profile of each batch of LMWH was determined using HPLC. To analyze in vitro activities, LMWHs were supplemented to normal human plasma and assessed using amidolytic anti-FXa and anti-FIIa, fibrinokinetic, and thrombin generation assays. Human whole blood was supplemented with LMWHs and the dynamic blood coagulation process was analyzed by thrombelastography (TEG). Whole blood flow cytometry was used to assess the ability of the LMWHs to inhibit tissue factor (TF)-induced platelet activation and lipopolysaccharide (LPS)-induced neutrophil activation. To assess the in vivo effect of the LMWHs, primates treated subcutaneously with a dose of 1 mg/kg LMWH had blood samples drawn pre-treatment and at 4, 6, 12 and 24 hours. Ex vivo pharmacodynamic activities of TFPI release, TAFI inhibition, and thrombin generation inhibition were evaluated. Results: Molecular weight parameters and IC50 values for FXa and FIIa inhibition by branded and generic enoxaparin were comparable. In the in vitro thrombin generation and fibrinokinetic assays, branded enoxaparin exhibited a more potent anticoagulant effect demonstrating slower clot formation with a weaker final clot structure (p=0.01) than generic enoxaparin. Although both the branded and generic enoxaparin produced a concentration-dependent anticoagulant effect in the TEG, there was greater degree of variability for the generic product between blood donors and between batches resulting in a less predictable linear response as drug concentration increased. When the increase in TEG R-time was plotted vs. concentration, branded enoxaparin showed a stronger anticoagulant effect (p=0.05). A concentration-dependent reduction in TF-induced platelet P-selectin expression was observed with branded and generic enoxaparin producing a similar effect. Incubation of whole blood with LPS resulted in a dramatic increase in neutrophil CD11b expression (MFI: 13.1±2.8 vs. 249.0±42.1) which was reduced by increasing concentrations of LMWH. This effect appeared to be stronger for generic than branded enoxaparin (MFI: 165.2±31.9 vs. 208.9±25.9). In primates treated with branded and generic enoxaparin, anti-FXa activity assessed by AUC 0–24hrs was similar. Anti-FIIa activity, however, was significantly higher in primates treated with generic enoxaparin (135±28 vs. 91±20 (μg*hr)/ml; p=0.023). AUC for thrombin generation inhibition was (B) 932±59 vs. (G) 775±119 %inhibition*hr; p=0.029. AUC for TFPI release was (B) 1101±98 vs. (G) 822±13 (ng/*hr)/ml; p=0.006. AUC for inhibition of TAFI activation was (B) 780±73 vs. 906±69 % inhibition*hr; p=0.023. Conclusions: This investigation demonstrated a wider variation in anticoagulant response to generic enoxaparin in comparison to branded enoxaparin. This variation was due to the response of the individual subject as well as to the batch of the product. In addition, both in vitro and in vivo/ex vivo activity differences were observed between branded and generic enoxaparin in several parameters relevant to the antithrombotic effect of LMWH. These findings suggest that simple analytical characterization can establish good quality control in manufacturing but may not assure similarity in biological performance between branded and generic enoxaparin. Thus beside the routinely required characterization, inclusion of additional tests for biologic activities and pharmacodynamic profiling of generic products in animal models may provide useful information on the bioequivalence of the generic versions of enoxaparin. Disclosures: Walenga: Sanofi-Aventis, Paris, France: Research Funding. Jeske:Sanofi-Aventis, Paris, France: Research Funding. Hoppensteadt:Sanofi-Aventis, Paris, France: Research Funding. Cunanan:Sanofi-Aventis, Paris, France: Research Funding. Escalante:Sanofi-Aventis, Paris, France: Research Funding. Khan:Sanofi-Aventis, Paris, France: Research Funding. Bailey:Sanofi-Aventis, Paris, France: Research Funding. Fareed:Sanofi-Aventis, Paris, France: Research Funding. Bakhos:Sanofi-Aventis, Paris, France: Research Funding.

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A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

10.1055/s-0039-1687438 ◽

1979 ◽

Author(s):

A.S. Bhargava ◽

J. Heinick ◽

Chr. Schöbel ◽

P. Günzel

Keyword(s):

Blood Clotting ◽

Anticoagulant Activity ◽

Factor Xa ◽

Thrombin Time ◽

Beagle Dogs ◽

Clotting Time ◽

Relative Potencies ◽

Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

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Consumption Of Viiirrag During Delayed Coagulation Process

10.1055/s-0038-1652743 ◽

1981 ◽

Author(s):

M Hada ◽

S Ikematsu ◽

M Fujimaki ◽

K Fukutake

Keyword(s):

Whole Blood ◽

Hemophilia A ◽

Negative Relationship ◽

Rabbit Serum ◽

Clotting Time ◽

Crossed Immunoelectrophoresis ◽

Qualitative Assay ◽

Similar Quantity

Coagulational functions of VIIIR:AG are still unknown, while its serological significance in laboratory medicine has been established. In this study it will be analyzed the mechanism of the consumption of VIIIR:AG during the process of prolonged blood coagulation.Quantitative assay of VIIIR:AG in plasma and serum is measured by Laurell’s method using 1% agarose with anti-F.VIII rabbit serum and qualitative assay of VIIIR:AG is performed by crossed-immunoelectrophoresis with anti F.VIII rabbit serum.In 28 patients with Hemophilia A a negative relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and prolongation of PTT is estimated and also a positive relationship between serum VIIIR:AG / plasma VIIIR:AG ratio and serum VIII:vW / plasma VIII:vW ratio is obtained. However, serum VIIIR:AG shows the similar quantity to plasma VIIIR:AG in the cases within normal clotting time, which have been treated by the in vitro addition of thrombin or by transfusion of AHG in vivo. When heparin or synthetic antithrombin ( MCI-9038 ) is added into normal whole blood or F.XIII deficient whole blood, the case of normal whole blood shows delayed clotting time and decrease of serum VIIIR:AG, but the case of F.XIII deficient whole blood indicates no decrease of serum VIIIR:AG. Furthermore, in those conditions change of the concentration of CIg in serum, which has been pointed out with fibrin crosslinking, indicate the similar behavior as serum VIIIR:AG.The results obtained above might suggest that serum VIIIR:AG tend to decrease in the cases with prolonged clotting time, and the F.XIII activity might be involved in the consumption mechanism of serum VIIIR:AG in such an abnormal condition, as fibrin crosslinkage could not carry out properly.

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Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637

Thrombosis and Haemostasis ◽

10.1160/th08-09-0586 ◽

2009 ◽

Vol 101 (06) ◽

pp. 1051-1059 ◽

Cited By ~ 25

Author(s):

Christer Mattsson ◽

Tord Inghardt ◽

Margareta Elg ◽

Johanna Deinum

Keyword(s):

Competitive Inhibitor ◽

Direct Thrombin Inhibitor ◽

Anticoagulant Effect ◽

Thrombin Inhibitor ◽

Thrombin Time ◽

Clotting Time ◽

Binding Studies ◽

Plasma Coagulation ◽

Coagulation Assays

SummaryAZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochemical methods were used to assess and measure the biochemical and pharmacological properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilised α-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition constant K i = 2–4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clot-bound thrombin) or to thrombomodulin. The total amount of free thrombin generated in platelet-poor clotting plasma was inhibited concentration-dependently by AR-H067637, with a concentration giving half maximal inhibition (IC50) of 0.6 μM. Moreover, AR-H067637 is, with the exception of trypsin, a se-lective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found. AR-H067637 prolonged the clotting time concentration-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC50 93 and 220 nM, respectively). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein IIb/IIIa exposure, IC50 8.4 nM) and aggregation (IC50 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of α-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.

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Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029620960820 ◽

2020 ◽

Vol 26 ◽

pp. 107602962096082

Author(s):

Dalia Qneibi ◽

Eduardo Ramacciotti ◽

Ariane Scarlatelli Macedo ◽

Roberto Augusto Caffaro ◽

Leandro Barile Agati ◽

...

Keyword(s):

Molecular Weight ◽

High Performance ◽

Thrombin Generation ◽

Area Under The Curve ◽

Factor Xa ◽

In Vivo Studies ◽

In Vitro Tests ◽

Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

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Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin

Blood ◽

10.1182/blood.v118.21.1251.1251 ◽

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.

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Effect of New Synthetic Heparin Mimetics on Whole Blood Thrombin Generation In Vivo and In Vitro in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612979 ◽

2002 ◽

Vol 87 (02) ◽

pp. 238-244 ◽

Cited By ~ 8

Author(s):

J.P. Hérault ◽

A. Bernat ◽

C. Gaich ◽

J.M. Herbert

Keyword(s):

Venous Thrombosis ◽

Charge Density ◽

Whole Blood ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Lag Time ◽

Drug Candidate ◽

Platelet Factor

SummaryThe effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat.TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition.The effect of hirudin, the synthetic pentasaccharide SR90107/ Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin.Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.

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Factor Xa Inhibitory Profile of Apixaban, Betrixaban, Edoxaban and Rivaroxaban Does Not Fully Reflect Their Biologic Spectrum

Blood ◽

10.1182/blood-2018-99-116993 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2520-2520

Author(s):

Fakiha Siddiqui ◽

Siddharth Mehrotra ◽

Vishnu Venkitasubramony ◽

Rithik Raina ◽

Lorenzo Storino Ramacciotti ◽

...

Keyword(s):

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Kinetic Method ◽

Factor Xa ◽

Biochemical Properties ◽

Therapeutic Effects ◽

Distinct Individual

Abstract Introduction: There are four oral anti-Xa drugs currently available for clinical use in various indications. These drugs are claimed to mediate their therapeutic effects by solely targeting factor Xa. While these agents are structurally similar, their biochemical properties and their effects on blood coagulation differ. Such differences may impact their safety and efficacy profile. The purpose of this study was to demonstrate the differences among factor Xa inhibitors in terms of their in vitro anticoagulant activity and other biochemical effects. Materials and Methods: Commercially obtained powdered forms of Apixaban, Betrixaban, Edoxaban and Rivaroxaban were profiled in this study. Stock solutions of each drug were prepared at 1mg/ml. To investigate the effect on the whole blood clotting profile, thromboelastographic studies were carried out over a concentration range of 0.5 - 2.5 ug/ml and whole blood activated clotting time (ACT) was measured at 1.0 and 2.5 ug/ml. The anticoagulant profile in citrated human pool plasma was measured at concentrations of 0.062-1.0 ug/ml using such tests as prothrombin time (PT) and activated partial thromboplastin time (aPTT). The anti-Xa effects of each agent were measured using a kinetic amydolytic method. The inhibitory potency was calculated in terms of IC-50. Thrombin generation inhibition studies on each drug were carried out in human pool plasma in a concentration range of 0.0-1.0 ug/ml using calibrated aotomated thrombogram (CAT) assay (Diagnostica Stago, Paris, France). Fibrinokinetics studies were carried out using an optical kinetic method, where thrombin was used to trigger clot formation. All results were compiled in terms of mean + 1 SD of 3-5 replicates. Results: All of the anti-Xa agents produced concentration and assay-dependent effects in these studies. The summary of each agent's effects at selected fixed concentrations and the IC-50 of the anti-Xa activity is given in the Table. In the whole blood ACT at 2.5ug/ml, Edoxaban showed the strongest anti-coagulant effects followed by Rivaroxaban > Betrixaban, whereas Apixaban showed minimal effects. In the TEG analysis at 1ug/ml, Edoxaban exhibited stronger anti-coagulant effects as measured by various TEG parameters, including R-time, K-time, alpha, and MA. Edoxaban and Rivaroxaban showed comparable effects followed by Betrixaban, whereas Apixaban exhibited weaker effects. In the PT assay at 1ug/ml, Edoxaban showed stronger effects, whereas Apixaban, Betrixaban and Rivaroxaban were comparable. aPTT at 1ug/ml revealed that Edoxaban was the strongest anti-Xa inhibitor followed by Betrixaban, whereas Apixaban and Rivaroxaban were comparable. In the anti-Xa assay Edoxaban was stronger (IC-50 = 340ng/ml, 0.62uM) than Apixaban (IC-50 =400ng, 0.87uM), Rivaroxaban (IC-50 = 840ng, 1.9uM) and Betrixaban (IC = >1000ng, >2.22 uM). In the thrombin generation assays at 1ug/ml, Apixaban showed the strongest inhibitory activity (IC-50 = 50ng/ml, 108nm) followed by Edoxaban (IC-50 = 58ng/ml, 108nm), Betrixaban (IC-50 = 60ngml, 133nm) while Rivaroxaban showed relatively weaker activity (IC-50 = 100ng/ml, 299nm). In the fibrinokinetics study at 1ug/ml, the anti-Xa agents produced varying degrees of inhibition with Rivaroxaban (67%), Edoxaban (42%), Apixaban (32%) and Betrixaban (12%). Summary and Conclusion: These results demonstrate that the measured anti-Xa activity alone does not fully reflect the overall biologic spectrum of these agents. Assay dependent variations are exhibited by each of these drugs, revealing distinct individual profiles. Edoxaban was the only anti-Xa agent which consistently exhibited relatively stronger inhibitory profile which was proportional to its anti-Xa activity. These studies indicate that the oral anti-Xa drugs may modulate the hemostatic system through additional mechanisms independent of the inhibition of factor Xa. Table. Table. Disclosures No relevant conflicts of interest to declare.

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