Assay Dependent Variations in the Anticoagulant and Protamine Sulfate Neutralization Profiles of Generic Copies of Enoxaparin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 908-908 ◽  
Author(s):  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
Paul D. Ackerman ◽  
Debra A. Hoppensteadt ◽  
Curtis Vandenberg ◽  
...  
Keyword(s):  
Whole Blood ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Inhibitory Effects ◽  
Clotting Time ◽  
Fda Approval ◽  
Potential Safety ◽  
Branded Product ◽  
Generic Products ◽  
The Individual

Abstract Currently several generic versions of a branded low molecular weight heparin (LMWH; enoxaparin, Sanofi-Aventis, Paris, France) have become available for clinical use in several countries. Such products include Cutenox (Gland Pharma, Hyderabad, India), Dripanina (Ariston, São Paulo, Brazil) and Clenox (Pharmayect, Barranquilla, Columbia). Although these products are not currently approved for use in the U.S., several other manufacturers have sought FDA approval for their products. Due to a lack of specifications and guidelines, this approval is still pending. In order to compare the relative potency of different anti-factor Xa U/ml adjusted generic preparations, studies were designed to compare each of the individual generic LMWHs with the branded product. Additionally, multiple batches of some of the generic products were also profiled. All of the agents were tested in human whole blood and citrated plasma over a concentration range of 0.15 to 10 U/ml. Whole blood activated clotting time (ACT) and thrombelastography (TEG) measurements along with fibrinopeptide A (FPA) generation were compared. The plasmatic tests included anti-Xa and anti-IIa activity by amidolytic assay and aPTT, Heptest and PiCT clotting time assays. In addition, protamine sulfate neutralization profiles for these agents were investigated at fixed protamine concentrations of 12.5 and 25 μg/ml. In the whole blood assays, at concentrations < 2.5 U/ml, no significant differences were observed between the branded and potency adjusted generic LMWHs. However, in the plasma-based systems, assay-dependent variations were observed which were more obvious at concentrations > 1.25 U/ml (aPTT, anti-IIa, anti-Xa; p<0.05). Similarly, product and assay based variations were also observed in the protamine neutralization profile of these LMWHs. Moreover, marked differences in some assays were observed when different batches of the generic copies of LMWHs were tested. Additional studies carried out to profile the oligosaccharide composition also showed product and batch-dependent variations. The relative amounts of antithrombin affinity components among the different generic products and within product batches also exhibited some variations. These studies clearly demonstrate that some of the generic copies of enoxaparin may not produce comparable anticoagulant and thrombin generation inhibitory effects at anti-Xa potency adjusted doses. Such differences may not be clinically relevant in the prophylactic indications (dosages ≤ 40 mg O.D). However, in therapeutic or interventional indications (IV or SC dosage > 100 mg), these products may exhibit differential safety/efficacy profiles. These observations underscore the importance of clear guidelines on the chemical and biologic specifications for the acceptance of generic versions of LMWHs. Such measures are crucial to avoid any potential safety/inefficacy issues particulatly in indications where these drugs are used at higher dosages.

scholarly journals Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values

2019 ◽  
Vol 25 ◽  
pp. 107602961986349 ◽  
Author(s):  
Fakiha Siddiqui ◽  
Alfonso Tafur ◽  
Lorenzo Storino Ramacciotti ◽  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Inhibitory Effects ◽  
Clotting Time ◽  
Reversal Effect ◽  
Normal Human ◽  
Fxa Inhibitor ◽  
Andexanet Alfa

Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed to neutralize FXa inhibitors. We studied the reversal effect of AA for these inhibitors in various anticoagulant and thrombin generation (TG) assays. Individual aliquots of normal human plasma containing 1 µg/mL of apixaban, betrixaban, edoxaban, and rivaroxaban, were supplemented with saline or AA at a concentration of 100 µg/mL. Clotting profiles include prothrombinase-induced clotting time, activated partial thromboplastin time, and prothrombin time. Factor Xa activity was measured using an amidolytic method. Thrombin generation was measured using a calibrated automated thrombogram. Differential neutralization of all 4 anticoagulants was noted in the activated clotting time and other clotting tests. The FXa activity reversal profile varied with an observed decrease in apixaban (22%), betrixaban (56%), edoxaban (28%), and rivaroxaban (49%). Andexanet alfa also led to an increased TG in comparison to saline. The peak thrombin was higher (40%), area under the curve (AUC) increased (15%), whereas the lag time (LT) decreased (17%). Andexanet alfa added at 100 µg/mL to various FXa supplemented systems resulted in reversal of the inhibitory effects, restoring the TG profile; AUC, LT, and peak thrombin levels were comparable to those of unsupplemented samples. Andexanet alfa is capable of reversing anti-Xa activity of different oral FXa inhibitors but overshoots thrombogenesis in both the saline and FXa inhibitor supplemented systems. The degree of neutralization of Xa inhibitor is specific to each agent.

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 (<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.

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.

scholarly journals Differential Neutralization of Apixaban, Betrixaban, Edoxaban, and Rivaroxaban By Andexanet Alfa As Measured By Whole Blood Thromboelastographic Analysis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1155-1155
Author(s):  
Siddharth Mehrotra ◽  
Debra Hoppensteadt ◽  
Walter Jeske ◽  
Omer Iqbal ◽  
Alfonso J Tafur ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Surrogate Marker ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Saline Control ◽  
Unequal Variances ◽  
Healthy Donors ◽  
Andexanet Alfa ◽  
The Individual

Introduction/Background: Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed as an antidote to neutralize the bleeding effects of oral FXa inhibitors such as, Apixaban and Rivaroxaban. The relative biological effect of these drugs have been investigated using various clot based and amidolytic methods for FXa inhibition. This Factor Xa inhibitory activity acts as a surrogate marker for the circulating level of these agents. We have recently reported that the FXa activity of these Anti-Xa agents does not fully reflect their biologic spectrum (JCath 25,1-11,2019). Whole blood assays such as thromboelastographic analysis represent a global assay which takes into account both the plasmatic and cellular components of blood and provides a more physiologic endpoint to study the anticoagulant effects of these drugs. The purpose of this study was to investigate the anticoagulant effects of currently available oral Anti-Xa agents such as Apixaban, Betrixaban, Edoxaban, and Rivaroxaban and their relative neutralization by AA in terms of such thromboelastographic parameters as R, K, Angle and MA. Materials and Methods: Analysis was carried out in whole blood using thromboelastography (TEG) using the TEG 5000 Hemostasis System (Haemonetics Corp, Massachusetts). Blood was drawn from healthy donors in individual groups (n=5-10) into 3.2% citrated tubes. In the TEG cup for testing, 0.2 M CaCl2, saline (with a filler and control), each of the individual FXa Inhibitors at a final concentration(FC) of 1 ug/mL, AA at FC of both 100 ug/mL and 50 ug/mL were tested for the relative neutralization of the anticoagulant effects. TEG parameters such as R-time, K-time, angle and MA were measured. All results were compiled individually for the saline control, 100 ug/mL AA and 50 ug/mL AA supplemented systems. Statistical analysis was carried out via an F test for equality of variances followed by the appropriate t test for equal or unequal variances. Results: When comparing the anticoagulants directly to one another, it was observed that Edoxaban shows the strongest anticoagulant effects in both R and K time followed by Betrixaban, then Rivaroxaban which were very similar in their anticoagulative effects, with Apixaban showing the weakest anticoagulant effect as shown in Table 1A. In the reversal studies as shown in Table 1B, as measured by various TEG parameters, R-Time, AA (FC=100 ug/mL) showed full neutralization effects in Apixaban (p=.027), Betrixaban(p=<.01), Edoxaban(p<.01), and Rivaroxaban(p<.01). In K-time, Betrixaban and Edoxaban were fully neutralized (respectfully p=.049 and p=.035) with partial neutralizations of Apixaban and Rivaroxaban. No significant neutralization was noted in the Angle and MA. AA at 50 ug/mL showed full neutralizations, in R-time, in Betrixaban and Rivaroxaban (Betrixaban[p<.01] and Rivaroxaban[p=.0287]), AA at this concentration showed partial neutralizations of Apixaban and Edoxaban. In K-time, AA showed full neutralization of Betrixaban and Edoxaban (Betrixaban[p=<.01] and Edoxaban[p<.01]). Apixaban and Rivaroxaban saw no neutralization effect by AA at FC=50 ug/mL in K-time. AA did not exhibit any significant neutralization effects in the Angle or MA parameters. Summary and Conclusion: All of the 4 Agents produced measurable anticoagulative activities at 1 ug/mL as measured by the TEG parameters. Edoxaban exhibited the strongest anticoagulative effect followed by Betrixaban and Rivaroxaban whereas Apixaban showed much weaker anticoagulant effects. AA FC=100 ug/mL showed much stronger, consistent, and complete neutralization effects of all of the 4 FXa Inhibitors when compared to AA at FC=50 ug/mL. These results strongly suggest that regardless of the variable anticoagulative effect exhibited by the FXa Inhibitors, AA at FC=100ug/mL fully neutralized the anticoagulant effects of this agent as measured by the TEG parameters. AA is shown to be the most effective in neutralizing Betrixaban in R-Time and K-Time at both concentrations of AA. AA was seen to neutralize Apixaban the least. It can be concluded that the effect of AA as a neutralizing agent is both drug and donor dependent and therefore dosage adjustment may be needed for the optimal clinical outcome with this antidote. 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.

Measurement of whole blood factor Xa-activated clotting time during hemodialysis with low-molecular-weight heparin

1998 ◽  
Vol 12 (3) ◽  
Author(s):  
Masakazu Mori ◽  
Shigenori Yoshitake ◽  
Takaaki Kitano ◽  
Shunsuke Oda ◽  
Takayuki Noguchi
Keyword(s):  
Molecular Weight ◽  
Whole Blood ◽  
Low Molecular Weight Heparin ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Clotting Time ◽  
Activated Clotting Time

scholarly journals Prothrombinase Induced Clotting Time Is More Sensitive then aPTT and PT and Can be Used for the Monitoring of Anti-Xa Agents in Whole Blood and Plasma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3374-3374
Author(s):  
Debra Hoppensteadt ◽  
Emily Bontekoe ◽  
Fakiha Siddiqui ◽  
Ambar Farooqui ◽  
Omer Iqbal ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Anticoagulant Activity ◽  
Surrogate Marker ◽  
Factor Xa ◽  
High Sensitivity ◽  
Relative Sensitivity ◽  
Inhibitory Effect ◽  
Clotting Time ◽  
Higher Sensitivity

Introduction: Currently there are four commercially available oral anti-Xa agents namely apixaban, betrixaban, edoxaban and rivaroxaban available for indication specific clinical use. All agents used are at a fixed dosage and their use may be associated with potential hemorrhagic complications. Although factor Xa inhibitory effect is considered to be a surrogate marker for the biologic action of these drugs we have demonstrated that factor Xa inhibitory profile of apixaban, betrixaban, edoxaban, and rivaroxaban does not fully reflect their biologic spectrum (https://doi.org/10.1177/1076029619847524). Furthermore the prothrombin time (PT) and activated partial thromboplatin time (aPTT) methods are of limited sensitivity and are dependant on several other enodogenous factors. Prothrombinase induced clotting time (PiCT), (Pentaharm, Basel, Switzerland) is a sensitive test for the global monitoring of anticoagulant drugs including heparins and parenteral oral anti-Xa and anti-IIa agents. The test is based on the RVV venom activation of endogenous FVa which forms prothrombinase complex with phospholipids (PL), FXa and calcium. The clot-based end point is proportional to the activities of FXa and FIIa. This study is designed to compare the relative responses of PT, aPTT and PiCT test in normal human blood, retrieved plasma and agents supplemented plasma with the oral anti-Xa agents to demonstrate the relative sensitivity of this assay in comparison to the PT and aPTT. Materials and Methods: Active pharmaceutical ingredient versions of apixaban, betrixaban, edoxaban and rivaroxaban were obtained from commercial sources. All agents were prepared at a stock concentration of 100ug/ml in saline and diluted to a working solution of 10ug/ml. Serial dilutions were prepared in various matrices. Citrated plasma from normal individuals (n=50) was obtained from a commercial source (George King Biomedical, Overland Park, Kansas). Whole blood and plasma samples (n=20) were supplemented at a concentration of 0-1000 ng/ml. Whole blood samples were also centrifuged to obtain retrieved plasma. These samples were analyzed using a single stage PiCT, aPTT and PT. Whole blood and retrieved plasma studies were carried out by using ST4 (Diagnostica Stago). PiCT were measured by using Pefakit PiCT. aPTT measurements were made by using Tcoag, TriniCLOT aPTT (Diagnostica Stago, Paris, France). For PT, HemosIL TM (Instrumentation Laboratory, MA, USA) was used. All results were compiled in individual groups as mean + SD. Results: All of these drugs produced a concentration dependent anticoagulant effects in the PiCT, aPTT and PT tests in both the whole blood and plasma systems. PiCT consistently showed much higher sensitivity in comparison to other tests. When compared at 1000ng/ml the anticoagulant effects of these drugs were stronger in plasma systems as shown in the table. PiCT consistently showed higher sensitivity in comparison to PT and aPTT in the whole blood, retrieved plasma and agent supplemented plasma. Edoxaban showed the strongest anticoagulant activity measured by PiCT in comparison to the other agents. Matrix based variations in the PiCT results were observed. Interestingly, the retrieved plasma from whole blood showed weaker anticoagulant effects in comparison to the directly supplemented plasma systems. Discussion: In comparison to PT and aPTT, the PiCT test was found to be the most sensitive in the three matrices studied. In the whole blood and plasma-based systems PiCT test showed linearity and high sensitivity for all of the four anti-Xa agents. In the PiCT test, consistently drug supplemented plasma showed the highest response in comparison to retrieved plasma and whole blood, suggesting differential binding of these drugs to cells. These results indicate that the PiCT test can be reliably used for the monitoring of anti-Xa agents. PiCT test can also be performed on currently available optical and mechanical instrument used for clotting studies. Owing to rapid turnaround time, high sensitivity, and lower cost PiCT can be used for the routine monitoring of oral anti-Xa agents. Table 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.

Measurement of whole blood factor Xa-activated clotting time during hemodialysis with low-molecular-weight heparin

1998 ◽  
Vol 12 (3) ◽  
pp. 130-132 ◽  
Author(s):  
Masakazu Mori ◽  
Shigenori Yoshitake ◽  
Takaaki Kitano ◽  
Shunsuke Oda ◽  
Takayuki Noguchi
Keyword(s):  
Molecular Weight ◽  
Whole Blood ◽  
Low Molecular Weight Heparin ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Clotting Time ◽  
Activated Clotting Time

Potency Adjusted Generic Versions of Argatroban Can Be Differentiated from Branded Argatroban in Thrombin Generation and Platelet Activation Assays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4007-4007
Author(s):  
Cafer Adiguzel ◽  
Omer Iqbal ◽  
Michael Sammikannu ◽  
Josephine Cunanan ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Generic Product ◽  
Cellular Systems ◽  
Thrombin Time ◽  
Clotting Time ◽  
Activated Clotting Time ◽  
Branded Product ◽  
Generic Products

Abstract Argatroban represents a widely used direct parenteral thrombin inhibitor for the anticoagulation management of patients with heparin-induced thrombocytopenia. Several generic versions of argatroban namely Slovastan, Gartban and Argaran have also become available in Japan. Although the antithrombin potency of the generic products of argatroban is adjusted to be comparable to the branded product, apparent differences in the pharmacodynamic effects have been noted in thrombin generation and platelet activation assays. To further investigate the bioequivalence of the three generic products with the branded argatroban, these agents were compared in whole blood (WB), platelet rich plasma (PRP) platelet poor plasma (PPP) and isolated biochemical systems. In the WB assays, the activated clotting time (ACT) studies were carried out mimicking the anticoagulant dosing (0–5 ug/ml). In the citrated WB, PRP and PPP various clotting tests such as the prothrombin time/INR (PT/INR), activated partial thromboplastin time (APTT), Heptest, prothrombinase activated clotting time (PICT) and thrombin time were carried out. To test the effect of these agents on tissue factor mediated activation of blood cells, flow cytometric studies were carried out. In addition, thrombin generation markers such as the fibrinopeptide A, thrombin/antithrombin complex and prothrombin fragment 1.2 were also measured. The effect of different forms of argatroban were also investigated on Xa and thrombin generation inhibition. While there was no difference in the anticoagulant effects of the branded and generic products in the clotting assays such as the PT, APTT, Heptest, PICT and thrombin time, matrix based differences were apparent. In the ACT assay, the anticoagulant effect of the branded and generic product were approximately the same, however, upon supplementation of the tissue factor the relative anticoagulant effects of these agents differed. All of the agents also produced a concentration dependent inhibition of the generation of microparticles in the WB studies where each of these agents were differentiated. Argaran produce weaker responses than the other agents. All of the agents also blocked p-selectin expression induced by tissue factor with an IC50 ranging from 1.8–2.3 ug/ml. There were obvious differences among the generic and branded products. In the thrombin and Xa generation assays differences were also noted between the generic and branded product. The relative ability of the generic and the branded argatroban in inhibiting the activation of thrombin activatable fibrinolysis inhibitor (TAFI) showed noticeable differences. These studies clearly indicated that while in the antithrombin titration and global anticoagulant assays the generic brands of argatroban exhibit comparable effects, in cellular systems and other assays differences between the generic product and branded versions can be noted. These obvious differences may be related to the solution matrix and the relative proportion of different forms of argatroban. These observations warrant additional pharmacoequivalent studies on the generic product to assure clinical equivalence of these products.

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