N(α)-Boc-l-Asp(OBn)-l-Lys(Z)-OtBu (reversin 121, 1), an inhibitor of the P-gp ABC transporter, was used to conceive compounds inhibiting the drug efflux occurring through the Hoechst 33342 and daunorubicin transport sites of P-gp, respectively H and R sites. Replacement of the aspartyl residue by trans-4-hydroxy-l-proline (4(R)Hyp) gave compounds 11 and 15 characterized by half-maximal inhibitory concentrations (IC(50)) of 0.6 and 0.2 μM, which are 2- and 7-fold lower than that of the parent molecule. The difference in IC(50) between 11 and 15 rests on the carbonyl group of the peptidyl bond, reduced in 15. Those compounds are rather specific of P-gp, having no or limited activity on MRP1 and BCRP. 15 displayed no marked cytotoxicity up to 10-fold its IC(50). Importantly, 15 equally inhibited the Hoechst 33342 and daunorubicin effluxes through a typical noncompetitive inhibition mechanism, suggesting its binding to a site different from the H and R drug-transport sites.

EP224283 combines in a single molecule idraparinux and tirofiban, which allows obtaining a predictable and sustained antiplatelet effect through the transfer of the pharmacokinetics properties of idraparinux to the anti-␣IIb␤3 antagonist. The activity can be instantaneously neutralized by injection of avidin, a specific antidote. We have tested the effects of this new profile anticoagulant in various thrombosis models. The antithrombotic effect of EP224283 was compared with those of the parent compounds used alone or in association at doses achieving low to moderate inhibition of platelet aggregation ex vivo. In a model of systemic thromboembolism independent of thrombin generation, tirofiban and EP224283 had similar effects at equimolar doses. On the other hand, EP224283 was more potent than tirofiban or idraparinux under thrombin-dependent conditions. In a ferric chloride-induced thrombosis model, EP224283 was more potent than either parent compound or their combination. Similar results were obtained after atherosclerotic plaque rupture in ApoE(Ϫ/Ϫ) mice. Thus, the dual action of EP224283 exceeds that of the parent compounds used in combination. A possible explanation is that EP224283 could concentrate antithrombin inside the thrombus by binding to ␣IIb␤3 through the tirofiban moiety, as shown by immunolabeling of the occluded vessel. No prolongation of the bleeding time was observed at doses achieving strong antithrombotic effects, suggesting that low to moderate ␣IIb␤3 inhibition combined with factor Xa inhibition minimizes the bleeding risk. The favorable antithrombotic profile of EP224283 together with its possible neutralization by avidin makes it an interesting drug candidate for the treatment and prevention of acute ischemic events.

EP217609 is a new synthetic parenteral dual-action anticoagulant combining a direct thrombin inhibitor (α-NAPAP analog), an indirect factor Xa inhibitor (fondaparinux analog) and a biotin moiety allowing its neutralisation. EP217609 exhibited similar in vitro anticoagulant properties as its parent compounds. On the basis of dose-response curves, we identified low and moderate doses of EP217609 resulting in similar ex vivo prolongation of the APTT as α-NAPAP analog and comparable ex vivo anti-FXa activity as fondaparinux. The effects of EP217609 were compared to those of its parent compounds used alone or in combination in two models of experimental thrombosis induced by FeCl3 injury of the carotid artery or mechanical injury of atherosclerotic plaques in ApoE-deficient mice. When administered at low doses increasing the APTT by only 1.1 fold, EP217609 significantly reduced the thrombus area in both models as compared to α-NAPAP analog or fondaparinux alone, but not to the combination of these drugs. In contrast, at higher doses increasing the APTT 1.5 times, EP217609 was not superior to either parent compound. Low doses of EP217609 did not prolong the tail bleeding time or increase the volume of blood loss, although a tendency towards an increased blood loss was observed in five out of 12 mice. Finally, the effects of EP217609 could be neutralised in vivo by injection of avidin. The pharmacological profile of EP217609, its performance in arterial thrombosis models and its possible neutralisation make it an interesting molecule and a potential candidate as an antithrombotic drug.

Zeodration represents a new strategy to prepare PLTs with partly preserved aggregative properties after storage and rehydration. Z_PLTs have potential hemostatic properties provided it is possible to improve their transfusion efficacy.