Characteristics of High and Low Molecular Weight Heparin Chains 0.7 0.1 0.2 0.3 0.4 0.5 0.6 UNFRACTIONATED HEPARIN % ENOXAPARIN 5000 10000 15000 Standard unfractionated heparin is a heterogeneous mixture of heparin chains with molecular weights ranging from 5 to 30 thousand daltons. Low molecular weight heparins are a class of compounds obtained by fractionating or depolymerizing this mixture into chains which have average molecular weights below 8 thousand daltons. Enoxaparin has a mean molecular weight of 4500 daltons. Eighteen saccharides is the critical chain length which differentiates the low molecular weight chains. Chains longer than 18 saccharides (over 5400 daltons) exhibit both anti-IIa and anti-Xa activity, are sensitive to inactivation by platelet factor 4, bind non-specifically to plasma proteins and endothelial cells, and are less efficient at inhibiting the generation of thrombin. The lower molecular weight chains primarily inhibit factor Xa, are resistant to inactivation by PF4, are less bound to plasma proteins, and are efficient inhibitors of thrombin generation. 5400 18 saccharides Molecular Weight (daltons) Anti-Xa Resistant to PF4 Little non-specific binding Inhibition thrombin generation Anti-IIa and anti-Xa Sensitivity to PF4 Non-specific binding Less inhibition thrombin generation 2

Resistance to Neutralization by PF4 Comparison of Neutralization of UFH and Enoxaparin by PF4 in Three In Vitro Assays 100% 80% 60% 40% 20% 0% Anti-Xa Thrombin Generation AntiIIa % of neutralization Due to the platelet-rich nature of arterial thrombi, resistance of low molecular weight heparins to degradation by platelet factor 4 is probably also an important advantage of standard heparin in arterial thromboses. In this in vitro study of the ability of platelet factor 4 to neutralize anti-Xa, anti-IIa and inhibition of thrombin generation, the anti-Xa activity of unfractionated heparin (shown on the left) was 94% neutralized by platelet factor 4, whereas anti-Xa activity in the presence of enoxaparin on the right was only 18% neutralized. Similarly, the ability to inhibit thrombin generation (shown by the yellow bars) was almost completely neutralized in the case of heparin but only 40% neutralized with enoxaparin. As predicted, anti-IIa activity (shown by the orange bars) was neutralized by PF4 for both heparin and enoxaparin. Heparin Enoxaparin (From Padilla, et al. Br J Haematol 1992; 82:406 – 413) 3

Molecular Mass Distribution Comparison Between Enoxaparin and Dalteparin 4 6 8 10 12 2 ENOXAPARIN % DALTEPARIN Due to different methodology employed in the fractionation/depolymerization processes, the different low molecular weight heparins are not pharmacologically equivalent. They differ from each other in many important physicochemical properties such as their mean molecular weights, the molecular weight distribution of the chains, and the chemical structure of the ends of the chains. These variations confer important differences among the low molecular weight heparins with respect to the ratio of anti-Xa to anti-IIa activity, bioavailability when administered subcutaneously, elimination half-life, and interactions with platelets. Displayed on this slide are the molecular weight distributions of the two low molecular weight heparins that have been studied in unstable angina. Of the two, enoxaparin has the highest percentage of the low molecular weight fragments below 2,000 daltons, and dalteparin has the lowest percentage. Conversely, enoxaparin has the lowest percentage of high weight fragments above 8,000 daltons, and dalteparin has the highest percentage. 2000 5400 8000 molecular mass (da) ENOXAPARIN 17% 55% 21% 7% DALTEPARIN 1% 43% 38% 18% 0 - 2000 2000 - 5400 5400 - 8000 >8000 Percent of Fragments with Specified Molecular Mass 4

Comparison of Anti-Xa and Anti-IIa Activities of Unfractionated Heparin and LMWH Anti-Xa Anti-IIa (IU/mg) (IU/mg) Ratio UFH 193 193 1.0 Dalteparin 130 58 2.2 Enoxaparin 98 25 3.9 This slide shows the relative anti-factor Xa and anti-factor IIa activities of unfractionated heparin, dalteparin, and enoxaparin. There are obvious differences in the ratio of anti-Xa to IIa activity in these three drugs, with enoxaparin having the highest anti-Xa to IIa ratio. (Adapted from Padilla, et al. Br J Haematol 1992;82:406 – 413) 5

Plasma Anti-Xa Activity After Ascending Single Doses of Subcutaneous Enoxaparin in Healthy Volunteers (mean +/- SD) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 mg/kg Anti-Xa activity (IU/ml) This slide shows the anti-factor Xa activity for a 1 mg/kg dose of enoxaparin in healthy subjects. Anti-factor Xa activity is measurable as early as 15 minutes post subcutaneous injection and peaks at 3-4 hours post injection. By 30 minutes post injection, enoxaparin treatment achieves anti-Xa levels comparable to the anti-Xa levels seen with therapeutic unfractionated heparin. Anti-factor Xa levels are quite predictable and consistent from patient to patient treated with similar doses of enoxaparin. 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (h) 6

Plasma Anti-IIa Activity After Ascending Single Doses of Subcutaneous Enoxaparin in Healthy Volunteers (mean +/- SD) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1.0 mg/kg Anti-IIa activity (IU/ml) This slide shows the anti-factor IIa activity for 1 mg/kg dose of exoaparin. As noted earlier, there is very little anti-IIa activity at this dose of enxaparin. 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (h) 7

Steady-State Anti-Xa Levels in Patients with Unstable Angina Receiving 1.0 mg/kg SQ every 12 hours 1.6 Trough 1.2 1.1 Peak 1.0 anti-Xa(IU/ml) 0.8 0.6 The large patient-to-patient variability in the dose of standard intravenous heparin required for a therapeutic effect is largely the result of non-specific binding to plasma proteins, including the acute phase reactants present in patients with acute coronary syndromes. In contrast, a weight-adjusted dose of subcutaneous enoxaparin results in predictable anti-Xa levels. This slide shows the measured anti-Xa levels at peak and trough for patients with unstable angina. At steady state, anti-Xa levels of 1.1 anti-Xa units per milliliter at peak and 0.6 at trough are attained. By contrast, a continuous infusion of intravenous unfractionated heparin, adjusted to an activated PTT of 1.5 to 2.5 times control, generally results in anti-Xa inhibition of between 0.3 and 0.6 anti-Xa units per milliliter, and requires frequent blood monitoring and adjustment to maintain this anti-Xa range. 0.5 0.4 3rd dose Last dose 8

aPTT Following SC Injection of Enoxaparin (mean +/- SD) 25 30 35 40 45 50 55 60 1.0 mg/kg Clotting time (seconds) This slide shows the aPTT values for a 1mg/kg dose of subcutaneous enoxaparin, 1 mg/kg. As seen here, there are only modest changes in aPTT associated with this dose of enoxaparin. These aPTT changes are also quite predictable and consistent from patient to patient treated with similar doses. There is no need to monitor aPTT during enoxaparin therapy, as this does not provide any information that would affect patient management. 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (h) 9