Long-lasting enfuvirtide carrier pentasaccharide conjugates with potent anti-human immunodeficiency virus type 1 activity.

Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG(12)-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.

From heparin to EP217609: the long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile.

The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.

Enhancement of the biological activity of BMP-2 by synthetic dextran derivatives.

In the present study, we explored the binding capacity of synthetic heparin-like dextran derivatives to recombinant human bone morphogenetic protein 2 (BMP-2), a heparin-binding osteoinductive growth factor. Affinity electrophoresis analysis provided evidence that carboxymethylated dextran polymers grafted with high amounts of benzylamide groups (named DMCB) interact with BMP-2. The capability of such polysaccharides to potentiate the growth factor biological activity was then investigated. In vitro, DMCB dose-dependently promoted osteoblast differentiation induced by BMP-2 in C2C12 myoblasts more efficiently than heparin. A screening study provided evidence that the potentiating effects of the dextran derivatives on the BMP-2-induced alkaline phosphatase activity improved with their benzylamide groups content and, therefore, with their affinity for the growth factor. The biological activity of BMP-2 was monitored in the culture medium after 6 days using C2C12 cells (containing a BMP sensitive luciferase reporter gene). Like heparin, DMCB sustained the biological activity of the growth factor; this result suggests that the formation of the BMP-2/DMCB complex may protect the protein from being inactivated. In rats in vivo, DMCB also stimulated ectopic calcification mediated by BMP-2. These data indicate that dextran-based polysaccharides prolong the half-life of the growth factor and promote its biological activity.

Enhancement of PDGF-BB mitogenic activity on human dermal fibroblasts by biospecific dextran derivatives.

Dextran derivatives are biosynthetic polyanionic polymers which exert some of the heparin properties such as regulating the activity of several heparin-binding growth factors. Based on a reproducible synthetic procedure, we have synthesized a new generation of dextran derivatives named dextran methylcarboxylate benzylamide sulfate (DMCBSu). Here we investigated the ability of a library of well-characterized DMCBSu to interact with platelet-derived growth factor-BB (PDGF-BB), which has essential roles during wound healing. Using gel mobility shift assay, our results indicate that benzylamide and sulfate groups act synergically to bind to PDGF-BB. Furthermore we show that depending on their chemical composition, functionalized dextrans are able to potentiate the mitogenic activity of PDGF-BB on human dermal fibroblasts. This enhancing effect is accompanied with changes in PDGF-BB-induced signaling events, as determined by the use of specific inhibitors and by western blot. Our results suggest that the use of such biopolymers combined with a local administration of the growth factor could increase the efficiency of the biomolecule activity in future therapeutic strategies.

Polysaccharide microarrays for polysaccharide-platelet-derived-growth-factor interaction studies.

Polysaccharide microarrays have great potential for the high-throughput analysis of polysaccharide-protein interactions. Here we demonstrate that a polysaccharide microarray prepared by printing a library of dextran polymers derivatized by methylcarboxylate, benzylamide, and sulfate groups (DMCBSu compounds) on to glass slides permitted the rapid identification of a set of compounds able to interact with the platelet-derived growth factor BB (PDGF-BB) isoform, a growth factor involved in wound healing. Microarray interaction results were compared to the capacity of DMCBSu compounds to potentiate the in vitro PDGF-BB-induced proliferation of human dermal fibroblasts.

Concentration of amino acid neurotransmitters in the somatosensory cortex of the rat after surgical or functional deafferentation.

Hindlimb unloading is considered as a model of functional deafferentation, since in this situation the tactile information from the paw and the proprioceptive input from the limb are dramatically reduced. Unloading induces a shrinkage of the cortical representation of the affected body part associated to a reorganization of topographic maps and to an expansion of receptive fields. Previous studies have suggested that cortical plasticity was the result of a change in the balance of excitation and inhibition in the cortex. The aim of the present study was thus to determine whether deafferentation of the hindlimb representation in the somatosensory cortex, by 14 days of unloading or by surgical means (selective dorsal rhizotomy during 17 days), can change the concentration in various amino acid neurotransmitters in the deprived cortex. The present findings indicate that both types of deafferentation result in a decrease in inhibitory amino acids (GABA, taurine) without significant changes in the main excitatory amino acid (glutamate). In conclusion, the present results support the idea that cortical changes are more likely due to a release from inhibition than to an increased excitation.

Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus.

In the filamentous fungus Aspergillus fumigatus, the vast majority of the cell-wall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/beta-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-beta-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositol-anchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphate-repressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus.