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Unfractionated heparin (UFH) and low-molecular-weight heparins have up to now been the anticoagulants of choice when a rapid anticoagulant effect is required. In the last few years, oligosaccharide synthesis has made considerable advances, allowing the development of new antithrombotic drugs such as the pentasaccharides [1,2]. The first synthetic pentasaccharide used in clinical trials, fondaparinux, has been shown to be highly effective in preventing venous thrombo-embolism after orthopedic surgery [3].

Idraparinux (SanOrg 34006) is a new long-acting analog of the synthetic pentasaccharide sequence. It is a potent and selective antithrombin dependent inhibitor of human factor (F)Xa with a strong affinity for antithrombin. It shows antithrombotic properties in in vitro and in vivo preclinical models. Its pharmacokinetic profile allows a once-a-week dosing regimen [4].

During the clinical use of idraparinux in the treatment and prevention of venous thromboembolic events, switches between UFH and idraparinux are possible. As both UFH and idraparinux are antithrombin-dependent, and due to expected competition, it appeared worthwhile to evaluate the possible interaction of idraparinux when administered concomitantly with UFH in the usual coagulation tests.

Therefore, we assessed the impact of the concomitant presence of UFH and idraparinux on their in vitro effects on pharmacodynamic variables in coagulation tests in the presence or absence of either protamine chloride or heparinase I (both heparin reversal agents). Idraparinux concentrations (based on determination of anti-Xa activity) and anti-IIa activity were also investigated.

Idraparinux (batch no. 600041-001-20 mg mL−1) and Heparine Choay® (5000 IU mL−1 of sodium heparin) were obtained from Sanofi-Synthelabo (Montpellier, France). Protamine chloride was obtained from Sigma (St Quentin Fallavier, France). Heparinase I was a gift from Dr E. Cohen (Hemoscope, Chicago, IL, USA).

Normal pooled plasma was obtained by pooling the plasma from 12 healthy volunteers recruited according to a prespecified protocol at the Center d'Investigation Clinique (Hôpital Saint Louis, Paris, France).

UFH and idraparinux were diluted in NaCl 0.9% and added together to pooled normal plasma in order to obtain increasing final concentrations of UFH: 0; 0.3; 0.6; 1; 2; 5 IU mL−1 and of idraparinux: 0; 0.2; 0.6; 2; 6 µg mL−1. To each mixture of UFH + idraparinux, NaCl 0.9% or a constant amount of protamine chloride (final concentration 36 µg mL−1) or a constant amount of heparinase I (final concentration 300 mIU mL−1) was added. Three sets of samples were prepared and run separately on three different days.

Activated partial thromboplastin time (APTT) and prothrombin time (PT) were performed on an ACL6000 from Instrumentation Laboratory (Paris, France) using Instrumentation Laboratory reagents: IL TestTM PT-Fibrinogen HS which contains polybrene, an inhibitor of heparin, and IL TestTM aPTT Liquid silica. APTT was expressed in seconds and PT as percentage of a control plasma.

Idraparinux concentrations (based on the determination of anti-Xa activity) and anti-IIa activity were measured in duplicate, using amidolytic methods in presence of an excess of antithrombin.

Increasing concentrations of UFH induced an expected prolongation of APTT and a concentration-dependent decrease of PT.

A slight prolongation of APTT and a slight decrease of PT occurred with increasing concentrations of idraparinux and seemed to reach a ‘plateau’ at the highest concentrations (2 and 6 µg mL−1).

The prolongation of APTT induced by UFH was not affected by the lowest concentrations of idraparinux (0.2 and 0.6 µg mL−1), but it was reduced by the highest concentrations (2 µg mL−1 and especially 6 µg mL−1) (Fig. 1).

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Figure 1. Effect of the combination idraparinux + unfractionated heparin (UFH) on activated partial thromboplastin time (APTT). Mean of three experiments.

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The decrease in PT induced by high concentrations of UFH (2 and 5 IU mL−1) was also reduced by the highest concentration of idraparinux (6 µg mL−1).

When both idraparinux and UFH were present, measured plasma idraparinux concentrations were greater than expected from the known added concentration of idraparinux, due to the additional anti-Xa activity of UFH. The magnitude of the effect was dependent on the relative concentrations of the two compounds, being greatest at low concentrations of idraparinux.

Protamine chloride by itself, at the high concentration used in these experiments, interfered with both PT and APTT. It decreased PT and slightly increased APTT. Heparinase had no effect on PT and APTT.

While protamine chloride and heparinase did neutralize the effects of UFH, they did not neutralize the effects of idraparinux.

In this study we describe the potential in vitro interactions of idraparinux, a hypermethylated, long-acting synthetic pentasaccharide (possessing a structure close to that of fondaparinux sodium) with UFH in coagulation tests. We also tested the in vitro effect of heparinase I and protamine chloride on this potential interaction.

Idraparinux interacts with human antithrombin with a dissociation constant in the nanomolar range. This affinity for human antithrombin is more than 10 times higher than that observed for fondaparinux [4]. In our study, at the high, supratherapeutic concentrations of 2 and 6 µg mL−1 (equivalent to about 1.25 and 3.75 µm), idraparinux decreased the effects of UFH on coagulation tests. This might be due to the much higher affinity of idraparinux for antithrombin resulting in a competition of idraparinux with UFH for antithrombin. Since idraparinux binds antithrombin in an equimolar ratio, antithrombin is saturated when the molar concentration of idraparinux exceeds that of antithrombin (around 2 µm in a normal pooled plasma). It can be assumed that this in vitro interaction of idraparinux with UFH on coagulation tests would be reversed by the addition of exogenous antithrombin. Similar effects with fondaparinux have been described previously [5].

The competition for antithrombin observed when measuring APTT and PT in normal pooled plasma was not observed when anti-FIIa activity and idraparinux concentrations were measured. This discrepancy could be explained by the assay methodology, since these measurements were realized in presence of an excess of exogenous human antithrombin.

Heparinase I and protamine chloride are effective pharmacological compounds for neutralizing UFH in vitro. The present results confirm that protamine chloride can significantly prolong prothrombin time; this has been shown to be through inhibition of FVII activation [6]. Previous data have shown that heparinase I could inactivate pentasaccharide in vitro while it had no effect on idraparinux [7,8]. Our results confirm that neither protamine chloride nor heparinase I affect idraparinux concentrations.

In conclusion, our observations could be relevant in the case of clinically possible, yet unlikely, situations of concomitant presence of UFH and supratherapeutic concentrations of idraparinux. In this situation, APTT values may underestimate UFH concentrations while idraparinux concentrations can be overestimated by the presence of UFH, but reliably measured after in vitro addition of heparinase I. Since, in this study, the concentrations of idraparinux, shown to interfere with UFH-induced alterations of coagulation tests, were already higher than the expected therapeutic concentrations, further in vitro experiments are needed to define more precisely the lowest concentration of idraparinux which interferes with UFH.

References

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