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.

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.

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.

Synthesis of thrombin-inhibiting heparin mimetics without side effects.

Unwanted side effects of pharmacologically active compounds can usually be eliminated by structural modifications. But the complex heterogeneous structure of the polysaccharide heparin has limited this approach to fragmentation, leading to slightly better-tolerated heparin preparations of low molecular mass. Despite this improvement, heparin-induced thrombocytopaenia (HIT), related to an interaction with platelet factor 4 (PF4) and, to a lesser extent, haemorrhages, remain significant side effects of heparinotherapy. Breakthroughs in oligosaccharide chemistry made possible the total synthesis of the pentasaccharide antithrombin-binding site of heparin. This pentasaccharide represents a new family of potential antithrombotic drugs, devoid of thrombin inhibitory properties, and free of undesired interactions with blood and vessel components. To obtain more potent and well-tolerated antithrombotic drugs, we wished to synthesize heparin mimetics able to inhibit thrombin, that is, longer oligosaccharides. Like thrombin inhibition, undesired interactions are directly correlated to the charge and the size of the molecules, so we had to design structures that were able to discriminate between thrombin and other proteins, particularly PF4. Here we describe the use of multistep converging synthesis to obtain sulphated oligosaccharides that meet these requirements.

Synthesis of a 3-deoxy-L-iduronic acid containing heparin pentasaccharide to probe the conformation of the antithrombin III binding sequence.

We report in this work the total synthesis of a close analogue of the pentasaccharide active site of heparin, in which the L-iduronic acid residue has been deoxygenated at position three. 1H NMR studies demonstrated that, as anticipated, such a modification induces a shift of the conformational equilibrium toward 1C4 (contribution to the conformational equilibrium rises from 37% to 65%) and a substantial decrease of the affinity for antithrombin III (Kd 0.154 microM versus 0.050 microM).

Synthesis and pharmacological properties of a close analogue of an antithrombotic pentasaccharide (SR 90107A/ORG 31540).

The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal models of venous thrombosis. We describe here the preparation and the pharmacological properties of 34, an analogue of oligosaccharide 1 with the latter's N-sulfates being replaced by sulfate esters and hydroxyl groups being methylated. These structural modifications allow a simpler and more efficient synthesis of such anionic oligosaccharides. Affinity for human AT III, anti-factor Xa activity, ability to inhibit thrombin generation, antithrombotic activity in a rat model of venous thrombosis, and elimination half-life in the rat have been determined for 1 and 34. Surprisingly, introduction of O-sulfates in place of N-sulfates, and methylation of hydroxyl groups, contributes to reinforce the binding to AT III, resulting in an improved pharmacological profile.

L-iduronic acid derivatives as glycosyl donors.

O-[Methyl (2-O-acetyl-3-O-benzyl-4-O-levulinyl-alpha, and beta-L-idopyranosid)uronate] trichloroacetimidate and the corresponding n-pentenyl glycosides are efficient L-iduronic acid glycosyl donors. Both have been used for the high-yielding synthesis of basic disaccharide blocks which are useful for the subsequent synthesis of complex oligosaccharides related to heparin/heparan sulfate, and dermatan sulfate. In contrast, the corresponding thioethyl glycosides, thiophenyl glycosides, and fluoride, did not yield the expected disaccharides.

Binding of heparin to antithrombin III: a chemical proof of the critical role played by a 3-sulfated 2-amino-2-deoxy-D-glucose residue.

Known methyl (prop-1-enyl 2,3-di-O-benzyl-alpha-D-glucopyranosid)uronate was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-levulinyl-alpha-D-gluco-pyranosid)uro nat e. Acid hydrolysis, followed by treatment with (bromomethylene)-dimethylammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-levulinyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose gave 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O- levulinyl-beta-D-glucopyranosyluronate)-beta-D-glucopyranose. Acetolysis, followed by selective anomeric O-deacetylation and treatment with (bromomethylene)dimethylammonium bromide then gave 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-levulinyl -beta-D-glucopyranosyluronate)-alpha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-4- O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-alpha-D- glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-levulinyl-beta-D- glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy - alpha-D-glucopyranosyl)- (1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)- 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glu copyranoside. Removal of the levulinyl group followed by condensation with 6-O-acetyl-2-azido-3,4-di-O -benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2- azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di- O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3- O- benzyl-2- deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-(1----4)-6-O-acetyl-3-O-benzyl-2-benzyloxycarbon ylamino-2- deoxy-alpha-D-glucopyranoside in 78% yield. O-Deacetylation followed by re-esterification, O-sulfation, catalytic hydrogenolysis, saponification, and N-sulfation gave the non-sodium salt of O-(2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl)-(1----4) -O- (beta-D-glucopyranosyluronic acid)-(1----4)O-(2-deoxy-6-O-sulfo-2-sulfoamino- alpha-D-glucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)- (1----4)-2-deoxy-6-O-sulfo-2-sulfoamino-D-glucopyranose. This synthetic pentasaccharide neither binds to antithrombin III nor induces anti-factor Xa activity.

Synthesis of heparin fragments: a methyl alpha-pentaoside with high affinity for antithrombin III.

The synthesis is described of the methyl alpha-glycoside of the pentasaccharide which represents the sequence in heparin responsible for binding and activation of the anticoagulant protein Antithrombin III. It was obtained in a yield much better than that of the previously synthesised pentasaccharide and exhibited the same biological properties.

Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1-4 )-O-(beta-D-glucopyranosyluronic acid)-(1-4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glu copyranosyl)-(1-4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose decasodium salt, a heparin fragment having high affinity for antithrombin III.

Known allyl 4,6-O-benzylidene-alpha-D-glucopyranoside was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosid)-uronate. Acid hydrolysis, followed by treatment with (bromomethylene)dimethyl-ammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-bet a-D-glucopyranose. Acetolysis, followed by treatment with titanium tetrabromide, then gave 3,6-di-O-acetyl-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-alp ha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxy- carbonylamino-2-deoxy-4-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-alpha-D-glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-(1- ---4)-O-(3,6-di-O-acetyl- -2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-gluc opyranoside. O-Dechloroacetylation followed by condensation with 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)- (1----4)-O-(methyl 2,3-di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)- O-(3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(m ethyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glucopyranoside in 70% yield. O-Deacetylation followed by re-esterification, O-sulfation, saponification, catalytic hydrogenolysis, and N-sulfation gave the decasodium salt of O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)-(1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gl ucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic+ ++ acid)-(1----4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose. This synthetic pentasaccharide binds to antithrombin III with an association constant similar to that of high-affinity heparin and elicits a potent anti-factor Xa activity in plasma.