Remi Huynh

Anticoagulant properties of dextranmethylcarboxylate benzylamide sulfate (DMCBSu); a new generation of bioactive functionalized dextran.

Dextranmethylcarboxylate benzylamide sulfate (DMCBSu), a functionalized dextran, exhibits anticoagulant properties. Its synthesis involves three steps: a carboxymethylation with monochloroacetic acid in alkaline water-iso-propanol, a benzylamidification of some of the methylcarboxylate groups with benzylamine in the presence of a water soluble carbodiimide and a partial sulfation of the remaining hydroxyl groups with SO3-pyridine in dimethylformamide. This procedure yields reproducibly DMCBSu with degrees of substitution in methylcarboxylate (MC), benzylamide (B) and sulfate (Su) groups, respectively, up to 1.61, 0.35 and 1.5, each obtained in one step. For a degree of substitution of methylcarboxylate ca. 1, the presence of sulfate groups is absolutely necessary to confer anticoagulant activities to the samples. In addition, the anticoagulant ability is higher for derivatives bearing benzylamide groups. The anticoagulant ability of DMCBSu increases with the degree of sulfation, reaching 20% of heparin activity for a degree of substitution of Su groups about 1.3.

Carboxymethylation of dextran in aqueous alcohol as the first step of the preparation of derivatized dextrans

Carboxymethyldextrans (CMD) with a degree of substitution of carboxymethyl groups DS > 0.9 are precursors for the synthesis of derivatized dextrans termed CMDBS, which present heparin-like biological properties. Syntheses of CMD in water/organic solvent mixtures using monochloroacetic acid (MCA) and strong alkaline conditions allowed to obtain highly substituted CMD with good reproducibility and high yields. The influences of, respectively, individual concentrations of reactants, composition and temperature of reaction medium, and allowed reaction time have been examined. The most favorable conditions for dextran carboxymethylation were obtained with 3.8 M NaOH and a [MCA]/[dextran] ratio of 2.5, allowed to react at 60°C for 90 min, in a reaction medium consisting of a tert-butanol/water (alternatively isopropanol/water) 85 : 15 (v/v) mixture. A DS of about 1.0 was obtained in one step, as compared to a DS less than 0.6 when using aqueous conditions. Improvements on overall CMD substitution levels (up to DS of 1.47) were achieved by merely repeating the reaction i. e. using the CMD products of the first reaction as substrate in the second. Carboxymethyldextrane (CMD) mit einem Substitutionsgrad der Carboxymethylgruppen DS > 0.9 sind Vorstufen fur die Synthese von derivatisierten Dextranen (CMDBS) mit heparinahnlichen biologischen Eigenschaften. Die Herstellung von CMD in wasrigen organischen Losungsmitteln und in Gegenwart von Monochloressigsaure (MCA) unter stark alkalischen Bedingungen fuhrt mit guter Reproduzierbarkeit und hohen Ausbeuten zu hochsubstituierten CMD. Der Einflus der Konzentration der Reaktanden, der Zusammensetzung und Temperatur des Reaktionsmediums sowie der Reaktionszeit wurden untersucht. Die gunstigsten Bedingungen fur die Carboxymethylierung von Dextran zeigten sich mit 3.8 M NaOH, einem [MCA]/[Dextran]-Verhaltnis von 2.5, einer Reaktionstemperatur von 60°C und einer Reaktionsdauer von 90 min in einer 85 : 15 (v/v) tert-Butanol/Wasser (alternativ Isopropanol/Wasser)-Mischung als Reaktionsmedium. Ein Substitutionsgrad von etwa 1.0 wurde mit einem Reaktionsschritt erhalten, wahrend unter wasrigen Bedingungen ein DS von weniger als 0.6 resultierte. Verbesserungen des CMD-Substitutionsgrades (bis zu DS 1.47) wurden durch wiederholte Reaktion erzielt.

Sulphated polysaccharides derived from dextran: biomaterials for vascular therapy

CMDBS are synthetic dextran derivatives randomly substituted with carboxymethyl (CM), benzylamide (B), sulphonate and sulphate groups (S). Depending on their overall composition, these compounds are endowed with heparin-like properties such as anticoagulant activity. Indeed, some CMDBS with high CM and S contents delay blood coagulation, whilst some derivatized dextrans without significant anticoagulant capacity are potent antiproliferative agents for rat smooth muscle cells (SMCs) in vitro as well as heparin. The growth inhibition is dose dependent, reversible and nontoxic. This result is of prime interest for medical use because proliferation of vascular SMCs is postulated to be a key step in the pathogenesis of atherosclerosis or restenosis after vascular surgery such as angioplasty. By varying the overall composition in the different substituents, we have also prepared CMDBS exhibiting a stimulatory effect on the in vitro growth of human endothelial cells (EC). Heparin, under similar experimental conditions, slightly inhibited EC growth. The data indicate a synergistic role of all substituents grafted onto the dextran backbone without considering that any can be responsible alone for this effect. We conclude that a suitable distribution of CM, B and S groups on dextran can mimic heparin activity in terms of anticoagulant activity and antiproliferative capacity on SMC growth. Moreover, some CMDBS are also endowed with a stimulatory effect on EC growth. These properties confer great interest to these synthetic polysaccharides for vascular therapy.