Inhibition of lipopolysaccharide-induced TNF-alpha production by semisynthetic polymyxin-B conjugated dextran

Abstract

During episode of severe endotoxemia, concentrations of both lipopolysaccharide and its lipid A-core subfraction are liberated from gram-negative bacteria and become elevated within the systemic circulation. Lipid-A core is the most homogeneous and physiologically toxic segment of the lipopolysaccharide molecule. Polymyxin-B has profound binding avidity for the lipid A-core subfraction of lipopolysaccharide. The mechanism of this binding avidity involves the development of attractive forces between the cationic groups of polymyxin-B and the anionic groups of the lipid A-core moiety of lipopolysaccharide. Complementary attractive forces include hydrophobic interactions which additionally become established between the octylheptanoyl group of polymyxin-B and the saturated carbon chains of the lipid A-core moiety. This paper describes a method for the semisynthetic production of polymyxin-B conjugated dextran in the form of polymyxin-B.ABH.dextran applying the photoreactive crosslinking reagent azidobenzoyl hydrazide (ABH). Molecular design and development of a semisynthetic technique for the conjugation of polymyxin-B to purified dextran fractions was motivated by the pronounced nephrotoxicity associated with this cationic polypeptide antibiotic. Conjugation of polymyxin-B to a relatively large molecular weight carrier compound would increase the overall size of the complex to a degree sufficient to theoretically reduce clearance through glomerular filtration mechanisms. Attributes of such a large molecular weight polymyxin-B conjugated biopharmaceutical would include diminished levels of nephrotoxicity due to a reduction of renal tubular concentrations and a simultaneous prolongation of its intravascular half-life (t (1/2)) and pharmacokinetic profile. Lipopolysaccharide (LPS) binding avidity of polymyxin-B.ABH.dextran was verified by Dot-Blot analysis in conjunction with the application of fluorescein isothiocyanate conjugated E. coli (0.55:B5) LPS (FITC-LPS). Capacity of polymyxin-B.ABH.dextran conjugates to inhibit in vitro LIP-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed by the application of a tissue culture based biological assay system capable of detecting cytotoxicity mediated by this potent monokine. Semisynthetic conjugates of polymyxin-B.ABH.dextran conjugates (0.6 microns/mL), thereby providing cytoprotectivity (95%; p < or - 0.001. to WEHI 164 clone 13 cell populations relative to untreated reference controls. Since TNF-alpha is currently believed to be the principal endogenous mediator involved in the host's inflammatory response during episodes of endotoxemia, results from these investigations provide a scientific foundation for warranting the elevation of the in vivo efficacy of large molecular weight semisynthetic polymyxin-B conjugates. Results from these investigations may ultimately lead to the application of semisynthetic polymyxin-B.ABH.dextran as a model for the molecular design of semisynthetic production of alternative biopharmaceutical or pharmaceutical agents possessing prophylactic and/or therapeutic efficacy for the management of severe endotoxemia conditions.