Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

First in man study of EP217609, a new long-acting, neutralisable parenteral antithrombotic with a dual mechanism of action.

UNLABELLED: EP217609 is a parenteral antithrombotic compound combining in one molecule an indirect anti-factor Xa inhibitor, a direct thrombin active site inhibitor and a biotin moiety. AIMS: The aim of the study is to investigate the safety, pharmacokinetics and pharmacodynamics of single ascending intravenous doses of EP217609. METHODS: In this randomised double-blind placebo-controlled study, healthy male subjects were administered intravenously single ascending doses (1, 3 or 10 mg) of EP217609 or placebo. Each treatment group consisted of 10 subjects of whom 8 received EP217609 and 2 received placebo. RESULTS AND CONCLUSIONS: All doses of EP217609 were well tolerated. A total of five treatment-emergent adverse events were reported, all considered unrelated, but no bleedings or other significant adverse events occurred during this study. In both plasma and urine, there was a strong correlation between EP217609 concentrations as measured by anti-factor IIa and Xa specific bioassays indicating that the two pharmacological activities of EP217609 did not dissociate in vivo. EP217609 pharmacokinetics were dose proportional and characterised by a low clearance, a small volume of distribution and a terminal half-life of 20.4 h. The long half-life was reflected in long-lasting, dose-dependent effects on activated and ecarin clotting time, thrombin and prothrombin time, activated partial thromboplastin time, thrombin generation time and anti-factor Xa activity. Pharmacokinetic/pharmacodynamic modelling indicated that the concentration of EP217609 producing 50 % of the pharmacodynamic effect was 3400 and 2210 ng/mL for activated clotting time and anti-factor Xa activity, respectively. These results warranted further clinical development of EP217609. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • There is a limited number of neutralisable anticoagulants, particularly when rapid neutralisation is required. • Synthetic anti-Xa compounds have predictable pharmacokinetic profiles. However, problems with thrombin rebound remain because of the inability to inhibit clot-bound thrombin. WHAT THIS STUDY ADDS: • This manuscript provides a comprehensive investigation of the pharmacokinetics, pharmacodynamics and safety of EP217609, and the results were the basis of future clinical studies in both healthy subjects and patients. • The pharmacokinetic/pharmacodynamic modelling provided information for dose selection in such future studies.

Synthesis, radiolabeling with fluorine-18 and preliminary in vivo evaluation of a heparan sulphate mimetic as potent angiogenesis and heparanase inhibitor for cancer applications.

Heparan Sulfate (HS) mimetics are able to block crucial interactions of the components of the extracellular matrix in angiogenic processes and as such, represent a valuable class of original candidates for cancer therapy. Here we first report the synthesis and in vitro angiogenic inhibition properties of a conjugated, novel and rationally-designed octasaccharide-based HS mimetic. We also herein report its labeling with fluorine-18 and present the preliminary in vivo Positron Emission Tomography imaging data in rats. This constitutes one of the rare examples of labeling and in vivo evaluation of a synthetic, polysaccharide-based, macromolecule.

EP42675, a synthetic parenteral dual-action anticoagulant: pharmacokinetics, pharmacodynamics, and absence of interactions with antiplatelet drugs.

BACKGROUND: EP42675 is a first-in-class, synthetic, parenteral, anticoagulant combining in a single molecule a direct thrombin inhibitor and an indirect factor Xa(FXa) inhibitor. OBJECTIVES: To investigate the safety, pharmacokinetics, and pharmacodynamics of EP42675 and its interaction with aspirin, clopidogrel, and unfractionated heparin (UFH). SUBJECTS AND METHODS: In study 1, healthy male subjects were administered intravenously single-ascending doses (1-10 mg) of EP42675 or placebo. In study 2, healthy male subjects were administered intravenously a single dose of 5 mg EP42675 on day 1 followed by oral administration of aspirin (100 mg) and clopidogrel (75 mg) once daily from day 8 to 21. On day 15, a second dose of 5 mg EP42675 was administered, and subjects were then randomized to receive a single dose of UFH (30 or 60 IU kg(-1) ) or placebo. RESULTS AND CONCLUSIONS: Mild bleedings were the only drug-related adverse events. EP42675 pharmacokinetics were dose-proportional and characterized by a low clearance, a small volume of distribution, a long terminal half-life. EP42675 pharmacodynamics were characterized by a long-lasting, dose-dependent increase in activated clotting time, ecarin clotting time, thrombin time, anti-FXa activity, activated partial thromboplastin time, prothrombin time, and a decrease in endogenous thrombin potential, measured by a thrombin generation test. Dose-dependent additive effects were seen with UFH on coagulation tests. EP42675 had no additive effect on the inhibition of platelet aggregation induced by aspirin and clopidogrel. These results warrant further clinical development of this new class of anticoagulant.

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy.

OBJECTIVE: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long-lasting antithrombin (AT)-dependent anti-factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. METHODS: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. RESULTS: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K(d) < 1 nm) and showed a potent anti-FXa effect and inhibition of thrombin generation with IC(50) values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long-lasting anti-FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. CONCLUSIONS: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former's pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients.

Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb-IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.

The structure of synthetic oligosaccharides in relation to factor IXa inhibition.

We investigated the effect of various oligosaccharides (OS) on the inhibition of factor IXa by antithrombin (AT) in a purified system. The OS comprised the AT-binding pentasaccharide sequence prolonged by saccharide chains with various lengths and charges. We show that factor IXa inhibition depended on the molecular weight of the OS. Factor IXa was not inhibited by the AT-binding pentasaccharide alone, but was inhibited if it was prolonged by a sulphated dodecasaccharide at the non-reducing end. The overall charge was also important since factor IXa inhibition was negligible if the pentasaccharide was prolonged by a non-sulphated dodecasaccharide. Using compounds containing a non-sulphated spacer, we showed that the central part of the OS was not critical. This study therefore demonstrates that the minimal OS structure necessary for catalysing factor IXa inhibition by AT is close to that required for catalysing thrombin inhibition.

Pharmacokinetics of new synthetic heparin mimetics.

Pharmacokinetics of oligosaccharides displaying various affinities for antithrombin (AT) allowed us to show that there was a close relationship between the plasma half-life of these antithrombotic oligosaccharides and their affinity for AT. Recently, we have described new heparin mimetics comprising an AT binding domain and a thrombin binding domain separated by a neutral methylated saccharide sequence. These compounds displayed strong anti-Xa and antii-IIA activities and, in contrast to heparin, escaped neutralisation by platelet factor 4. The aim of this work was to compare the pharmacokinetics of several of these heparin mimetics in rats. These compounds differed by their length, charge and affinity for AT (AT-binding domain). The results obtained indicate that the prolongation of the AT binding domain did not modify significantly their affinities for AT. However, an increase in the number of charges leads to a decrease in the half-life. When a methylated spacer was added in order to minimise the non-specific interactions to other proteins, half-lives of the heparin mimetic were in the same range than that of the pentasaccharide used as an AT binding domain. In conclusion, this study shows the influence of the charge of the oligosaccharides on their pharmacokinetics and underlinesthe importance of minimising their non-specific binding to plasma proteins in order to obtain compounds with predictive pharmacokinetics.

Conformation of heparin pentasaccharide bound to antithrombin III.

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.

SR123781A, a synthetic heparin mimetic.

SR123781A, a synthetic hexadecasaccharide comprising an antithrombin (AT) binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained from glucose through a convergent synthesis. SR123781A showed high affinity for human AT (Kd = 58 +/- 22 nM) and was a potent catalyst of its inhibitory effect with regard to factor Xa (IC50) = 77 +/- 5 ng/ml - 297 +/- 13 U/mg) and thrombin (IC50 = 4.0 +/- 0.5 ng/ml - 150 +/- 30 U/mg). SR123781A which acted exclusively via AT (no effect via heparin cofactor II at a concentration of 6 microg/ml) inhibited thrombin generation occurring via both the extrinsic and intrinsic pathways in vitro in human plasma. SR123781A did not compete for 3H-heparin binding to PF4 and did not activate platelets in the presence of plasma from patients with heparin-induced thrombocytopenia. After intravenous or subcutaneous administration to rats, rabbits or baboons, SR123781A displayed prolonged anti-factor Xa and anti-factor IIa activity ex vivo. After intravenous injection to baboons, decreases of the anti-factor Xa and anti-thrombin activities were parallel and disappeared with the same pharmacodynamics. Intravenous administrations of SR123781A strongly inhibited thrombus formation in an experimental model of thromboplastin-induced venous thrombosis in rats with an ED50 value of 18 +/- 0.1 microg/kg (vs 77 +/- 3 microg/kg for heparin). SR123781A inhibited arterial thrombus formation induced on a silk thread in an arterio-venous shunt and in the vena cava (ED50 values of 225 +/- 10 and 27 +/- 8 microg/kg, respectively). Compared to standard and low molecular weight heparin and to presently used drugs, SR123781A exhibited a highly favourable antithrombotic/bleeding ratio therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Experimental proof for the structure of a thrombin-inhibiting heparin molecule.

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.

The effect of a reducing-end extension on pentasaccharide binding by antithrombin.

Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >/=0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.

Oligosaccharides corresponding to the regular sequence of heparin: chemical synthesis and interaction with FGF-2.

It has been proposed that oligosaccharides corresponding to the so-called regular region of heparin/heparan sulfate (HS) bind to fibroblast growth factor-2 (FGF-2). In order to explore the molecular basis of FGF/HS interaction, we describe here the chemical synthesis of a tetra and a hexasaccharide, prepared as methyl glycosides, corresponding to the regular sequence of heparin. The strategy relies on the efficient preparation of three building blocks: a seeding block, an elongating block and a capping block. The hexasaccharide inhibited the binding of FGF-2 on its receptor on human aorta vascular smooth muscle cells with an IC50 value (16+/-1.2 microg/mL) close to that of standard heparin (14.8+/-0.5 microg/mL) whereas the tetrasaccharide was much less potent (IC50 = 127+/-10.5 microg/mL). The hexasaccharide and heparin, inhibited in a dose-dependent manner FGF-2 (30 nM) induced proliferation (IC50 = 23.7+/-1.6 and 30.1+/-1.3 microg/mL, respectively). Under the same experimental conditions, the tetrasaccharide only slightly inhibited the mitogenic effect of FGF-2 (IC50 > 100 microg/mL).

A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.

Identification of a hexasaccharide sequence able to inhibit thrombin and suitable for 'polymerisation'.

Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide building block precursors. One of them, methyl(sodium 2,3-di-O-methyl-4-O- sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]2-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside, obtainable from a single disaccharide building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics able to inhibit the two coagulation factors thrombin and Factor Xa.

Assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition.

Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]n-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside have been synthesized from a single disaccharide precursor. All of them bind to and activate antithrombin. When n < or = 6 only Factor Xa inhibition is observed, whereas when n > 6 Factor Xa and thrombin are both inhibited in the presence of antithrombin. These results indicate that, in heparin, the sequence involved in antithrombin-catalyzed thrombin inhibition is a pentadeca- or a hexadecasaccharide.

[New antithrombotic oligosaccharides]. / Nouveaux oligosaccharides antithrombotiques.

In the early eighties, following breakthroughs in oligosaccharide chemistry, the total chemical synthesis of pentasaccharides has been achieved, representing the antithrombin binding domain of heparin (the active site). The selective inhibitors of coagulation factor Xa thus obtained represent a new type of antithrombotic drugs. In a further step, based on the knowledge of the mechanism of antithrombin activation by heparin, oligosaccharides (pentadeca- to eicosasaccharides), comprising an antithrombin binding domain prolonged by a thrombin binding domain, were designed and synthesised in the Sanofi group. These compounds inhibit both factor Xa and thrombin, in the presence of antithrombin. Endowed with the full anticoagulant activity of heparin but devoid of undesired non-specific interactions, particularly with platelet factor 4 (PF4), they might represent "the ideal heparin-like antithrombotic".

New synthetic heparin mimetics able to inhibit thrombin and factor Xa.

Synthetic pentadeca-, heptadeca- and nonadecasaccharides, comprising an antithrombin III (AT III) binding pentasaccharide prolonged at the non-reducing end by a thrombin binding domain have been obtained. The pentadecasaccharide is the shortest oligosaccharide able to catalyse thrombin inhibition by AT III. The nonadecasaccharide is a more potent thrombin inhibitor than standard heparin.

Synthetic oligosaccharides having various functional domains: potent and potentially safe heparin mimetics.

A synthetic heptadecasaccharide, comprising an antithrombin III binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained through a convergent synthesis. This compound displayed in vitro anticoagulant properties similar to that of standard heparin but, in contrast with heparin, escaped neutralization by platelet factor 4, a protein released by activated platelets.