Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia

Abstract

Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60-70 kDa) and/or clearance by the body"s components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol [PEG] is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here we utilized a natural vertebrate carbohydrate, heparosan [HEP], the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The heparosan polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the non-human primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several heparosanmodified human granulocyte-colony stimulating factor [G-CSF] conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55-or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or heparosan alone for 2 weeks did not cause heparosan-specific toxic effects in rats. Heparosan did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-heparosan IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

“…To confirm this hypothesis, we used the Stokes-Einstein relation to calculate the infinite dilution values for the hydrodynamic radius r ( H ) 0 from the infinite dilution diffusion coefficients ( D 0 ) for each condition ( Table 3 ). The r ( H ) 0 values range from 2.1 up to 4.0 nm, whereas the reported value is 2.0 nm [57] , which confirms the presence of larger species in our samples. This in turn impedes the correct determination of the diffusion interaction parameter k D .…”

A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

“…Quasi-monodisperse size-defined heparosan (Hep-b) and chondroitin (C0-b) with a biotin tag at their reducing ends were custom made for this study using synchronized, stoichiometrically controlled chemoenzymatic reactions. For HEP-b, a heparosan trisaccharide amine derivative was extended with recombinant MBP-PmHS1 synthase using UDP-GlcNAc and UDP-GlcA (Sigma) [48]. For C0-b, a HA tetrasaccharide amine [47] was extended with recombinant PmCS1-704 synthase [49]) using UDP-GalNAc and UDP-GlcA [50, 51].…”

Novel G‐CSF conjugated anionic globular dendrimer: Preparation and biological activity assessment

“…These researches were aimed to achieve more active molecules or longer lifespans. [9][10][11][12][13] Nanotechnology is a promising field to modify biological macromolecules that often yielded positive results. Among nanoparticles, dendrimers have unique properties such as a globular structure and most importantly polyvalent properties that make them suitable agents in medicinal chemistry.…”

“…These researches were aimed to achieve more active molecules or longer lifespans. 9 , 10 , 11 , 12 , 13 …”

“…Different variants of G‐CSF have been developed for therapeutic purposes through amino acid addition, or substitution, chemical modifications with natural and synthetic polymers, and development of fusion forms. These researches were aimed to achieve more active molecules or longer lifespans 9–13 …”