Abstract

1. Comparative Studies on the Anticoagulant, Antiprotease and Thrombin Generation Inhibition on Various Generic Versions of Low Molecular Weight Heparins, Enoxaparins. Christina Bolek, Class of 2013 Proviso Mathematics & Science Academy, Forest Park, IL Commercially Available Branded and Generic Enoxaparins Abstract Materials and Methods 1. Anticoagulant studies; The anticoagulant studies were carried out by supplementing the PPP and PRP with each of the drugs at a concentration of 0-10ug/ml. Methods such as activated partial thromboplastin time, heptest time and thrombin time were measured. The results were expressed in terms of seconds. The anticoagulant studies were carried out on an ACL centrifugal analyzer (Instrumentation Laboratory, Lexington, MA) 2. Amidolytic studies; The plasma samples used in the anticoagulant studies were also assayed for antiprotease activities using amidolytic anti-Xa and IIa methods. The aminolytic studies were also carried out on ACL centrifugal analyzer monitoring the delta A 405. 3. Protamine neutralization studies; The protamine neutralization studies were carried out in the anticoagulant and antiprotease systems. To carry out these studies, saline was used as a control in one set, whereas, a fixed amount of protamine (10ug/ml) was supplemented to the plasma containing each of these drugs at a concentration range of 0-10ug/ml. The neutralization profile was determined by plotting the concentration response curves. All methods are standard methods used in the hemostasis and thrombosis research laboratories of the Loyola University Medical Center, where this research project was carried out. All methods are validated and have been used for routine screening of heparins and low molecular weight heparins. Objectives: Heparin is a commonly used anticoagulant drug used for the management of thrombosis. Low molecular weight heparins (LMWH) are derived from heparin by digestion methods using chemical enzymatic techniques. Enoxaparin is a low molecular weight heparin, which is most commonly used in the U.S. More recently, beside the branded (Sanofi Aventis) several generic equivalent products have become available (Watson). Additional generic products have also been developed in China (C1 & C2). The purpose of this study was to compare the anticoagulant and anti-protease activities, which are usually used to evaluate the potency of each product. This study was designed to demonstrate the equivalence of the generic versions of enoxaparins in comparison to the branded product. Materials & Methods: Commercially available branded enoxaparin was obtained from Sanofi Aventis, whereas the generic product was obtained from Watson laboratories. Two Chinese generic products were obtained from Medefil Inc. Plasma samples were prepared from healthy volunteers in an approved IRB protocol and supplemented with three individual batches of four different versions of enoxaparin in a concentration range of 0-10ug/ml. Similarly, to investigate the neutralization by protamine sulfate, these supplemented plasma samples were neutralized with protamine at a fixed concentration of 10ug/ml. Frozen platelet rich plasma was also used to determine the effect of platelets (platelet factor 4) on the anticoagulant and anti-protease activities. The clotting assays included aPTT, Heptest and Thrombin Time and were measured using an automated centrifugal analyzer (ACL). Chromogenic substrate based methods were used to study the anti-Xa and IIa activities. Results: The results of each product (three batches) were tabulated in terms of group mean±SD. The results were plotted in terms of concentration curves and compared in the PPP, PRP and protamine neutralization of the PPP supplemented systems. All drugs produced concentration dependent increases in the anticoagulant activities, which were measurable using Heptest and aPTT. The thrombin time was also affected and variable results were obtained. In the anti-Xa/IIa assays, these drugs produced concentration dependent inhibition of both enzymes. The PT was least affected by these drugs. In comparison to the PPP, the PRP supplemented system showed lower activities in most assays. Protamine sulfate was found to neutralize the anticoagulant activities partially; however, in the thrombin time and anti-IIa assay there was a stronger effect. Conclusions: These studies show that the anticoagulant and anti-protease profiles of the generic and branded enoxaparins are comparable. Within the products, batch-to-batch variations were found; however, these were not remarkable. These studies suggest that the currently manufactured generic LMWHs are comparable in their anticoagulant and anti-protease profiles. Additional studies on targeted tests such as thrombin generation and biochemically defined systems are needed to further compare these observations. Discussion • In the aPTT, PT and Heptest assays, there were no differences in the anticoagulant profiles between these drugs, except for C1, which showed slightly higher anticoagulant activity. (Results of the PT and aPTT not shown) • In the thrombin time assays, the anticoagulant and neutralization profiles were variable. • The anti-Xa activities exhibited somewhat variable results, which were statistically insignificant. • Similarly, the anti-IIa activities were comparable prior to neutralization and showed some variations. • These results represented a mean of three batches per group of drugs. In addition, the chemical depolymerization may also result in some structural changes, which may inflict some differences. • Protamine sulfate was more effective than platelet factor 4 in neutralizing these agents. However, assay based variations in the neutralization profile were noted. • On a cumulative basis, these results do not demonstrate any major differences among these drugs’ anticoagulant activities. Similarly, their neutralization profile are comparable. Thus, besides minor differences, the generic and branded drugs are comparable. Results Chinese 1 (Old Medefils) Watsons Objectives Unfractionated heparin is a commonly used anticoagulant drug, used for the management of thrombosis. Most of the heparin preparations used in the US are derived from mammalian tissues, such as the porcine mucusa heparin. Low molecular weight heparins (LMWH) are derived from heparin by digestion methods using chemical enzymatic techniques. Enoxaparin is a low molecular weight heparin, which is most commonly used in the U.S. More recently, beside the branded (Sanofi Aventis) several generic equivalent products have become available (Watson). The purpose of this study is to compare the anticoagulant and anti-protease activities, which are usually used to evaluate the potency of each product. Using both the biochemical and clot based methods, these drugs were compared in standardized systems. Chinese 2 (New Medefils) Branded Enoxaparin Conclusion These studies show that the anticoagulant and anti-protease profiles of the generic and branded enoxaparins are comparable. Within the products, batch-to-batch variations were found; however, these were not remarkable. These studies suggest that the currently manufactured generic LMWHs are comparable in their anticoagulant and anti-protease profiles. Additional studies on targeted tests such as thrombin generation and biochemically defined systems are needed to further compare these observations. Acknowledgments The results of each product (three batches) were tabulated in terms of group mean±SD. The results were plotted in terms of concentration curves and compared in the PPP, PRP and protamine neutralization of the PPP supplemented systems. All drugs produced concentration dependent increases in the anticoagulant activities, which were measurable using Heptest and aPTT. The thrombin time was also affected and variable results were obtained. In the anti-Xa/IIa assays, these drugs produced concentration dependent inhibition of both enzymes. The PT was least affected by these drugs. In comparison to the PPP, the PRP supplemented system showed lower activities in most assays. Protamine sulfate was found to neutralize the anticoagulant activities partially; however, in the thrombin time and anti-IIa assay there was a stronger effect. These studies were carried out during a student research mentorship program of the Proviso Mathematics & Science Academy during the period of November 2nd, 2012 through April 9th, 2013. The research was conducted at the Hemostasis and Thrombosis Research Laboratories under the supervision of Dr. Jawed Fareed and Mr. Daneyal Syed, BS. I am thankful to Mme Delaney, my mentorship teacher, and Ms. Coriano, my counselor, for helping me and guiding me through my experiences.