Comparative Antithrombotic and Bleeding Effects of Two U.S. Generic Enoxaparins

Low molecular weight heparins (LMWHs) are complex biologic drugs that exhibit heterogeneity in terms of saccharide chain length and in the composition (sulfate, acetyl), content, and location of functional groups. Such heterogeneity impacts the biologic activity of LMWHs as there is a certain threshold chain length required for thrombin inhibitory activity and a particular sequence is required for interaction with antithrombin. In July 2010 the US Food and Drug Administration published requirements necessary to demonstrate the ‘sameness’ of generic LMWHs with the originator LMWH. We undertook this study to compare the primary anticoagulant effect of thrombosis prevention and the primary adverse effect of bleeding of two FDA approved generic enoxaparins.

Four batches of commercially available Sandoz US generic enoxaparin (Princeton, NJ) and two batches of Watson US generic enoxaparin (Parsippany, NJ) were compared. All products were obtained from hospital pharmacies as pre-filled syringes containing 40 mg of the drugs. The molecular weight profile of each batch was determined by HPLC in relation to well-defined heparin fractions and by utilizing the US Pharmacopeia method. In vitro activity was determined by supplementing each LMWH batch to normal human plasma over a range of concentrations (0–10 mg/ml) and analyzing these samples using aPTT, anti-FXa and anti-FIIa assays. Hemorrhagic activity was measured using a rat tail transection model five minutes after administration of a 2 mg/kg intravenous dose (n=8 rats/batch). Upon completion of the bleeding model, antithrombotic activity was assessed using a jugular vein clamping model (∼90 minutes post-dosing). Blood samples collected from treated rats were used to estimate circulating blood levels of LMWH using anti-FXa and anti-FIIa assays.

The two groups of generic enoxaparins exhibited a similar molecular weight profile with mean molecular weights of 4,270 ± 20 Da for the Sandoz products and 4,420 ± 80 Da for the Watson products. In vitro activities were similar between batches of the same product, but the individual products differed considerably (p=0.01). In the aPTT and anti-FIIa assays, the Watson LMWH produced significantly more activity than the Sandoz LMWH at concentrations ≥5 μg/ml. At 5 μg/ml, a clotting time of 74.0 ± 16.6 sec was observed with Watson batches compared to clotting times ranging from 52.9 to 54.9 sec for Sandoz LMWHs. While a similar pattern was observed with the anti-FXa assay, the differences between products were not statistically significant. In the bleeding model, all LMWHs prolonged the bleeding time compared to vehicle control. One batch of the Watson LMWH, however, produced a significantly longer bleeding time compared to all other samples tested (33.8 ± 5.1 min vs. a range of 11.9 ± 2.4 to 18.3 ± 4.3 minutes for the other samples; p=0.003). The same batch produced significantly more antithrombotic activity (6.3 ± 0.7 clampings) compared to the other samples (range of 4.0 ± 0.6 to 4.6 ± 0.5 clampings; p<0.001). One batch of Sandoz LMWH produced a significantly smaller prolongation of bleeding time compared to other samples. Circulating drug levels determined by anti-FXa and anti-FIIa activities were comparable in all treatment groups but did not appear to correlate with hemorrhagic or antithrombotic activities.

The expectation was that all batches of all generic enoxaparins would produce the same in vitro, in vivo, and ex vivo outcomes. The findings of this study suggest that incorporation of traditional animal models in the development of generic enoxaparins may be of value as multiple biological effects of LMWHs, not fully addressed with the anti-FXa and anti-FIIa activities, contribute to the overall antithrombotic activity of these LMWHs. This study demonstrates that all generic enoxaparins available today may not necessarily be the same. These findings underscore the importance of in vivo studies in animal models to demonstrate the bioequivalence of the generic products.

Jeske:Sanofi-Aventis, Paris, France: Research Funding. Walenga:Sanofi-Aventis, Paris, France: Research Funding. Escalante:Sanofi-Aventis, Paris, France: Research Funding. Hoppensteadt:Sanofi-Aventis, Paris, France: Research Funding. Cunanan:Sanofi-Aventis, Paris, France: Research Funding. Kahn:Sanofi-Aventis, Paris, France: Research Funding. Paulus:Sanofi-Aventis, Paris, France: Research Funding. Fareed:Sanofi-Aventis, Paris, France: Research Funding. Bakhos:Sanofi-Aventis, Paris, France: Research Funding.