The objective of the proposed work is to develop an inhibitor of anthrax toxin that is effective in vivo and that is suitable for clinical use. Anthrax toxin is a combination of three proteins that are secreted separately by Bacillus anthracis and form toxic complexes that are internalized by mammalian cells. Protective antigen (PA) binds cellular receptors and is cleaved by furin-like proteases, which leads to the heptamerization of the cell-associated PA63 fragment. Heptamerization allows binding of the enzymatic toxin components, edema factor and lethal factor, and triggers endocytosis of these complexes and intoxication of the cells. Since anthrax toxin is an essential virulence factor and is responsible for the major symptoms and death from anthrax, the toxin is a prime target for therapeutic intervention. In particular, PA is an ideal target because it is the common component of the two toxins that comprise anthrax toxin, edema toxin and lethal toxin, which both induce tissue damage and cause death. We have attached multiple copies of a PA6s heptamer-binding inhibitory peptide to both poly-L-glutamic acid (PLGA) and liposomal scaffolds to generate polyvalent molecules that are several orders of magnitude more potent that the monomeric peptide in vitro and that neutralize anthrax toxin in vivo. The advantages of the polyvalent inhibitors described in this proposal are: 1) they block both toxins; 2) they are synthesized from non-toxic scaffolds that are in routine clinical use (liposomes) or have been shown to be safe in clinical trials (PLGA); 3) the synthetic processes are facile and scaleable; 4) polyvalency provides a significant enhancement of potency; and 5) the PLGA-based inhibitors do not require a cold chain.
The first aim of this proposal is to optimize the in vivo efficacy of polyvalent anthrax toxin inhibitors. We will synthesize a limited number of PLGA-based and liposome-based inhibitors that exhibit high potencies in vitro and that differ in size (molecular weight of PLGA; diameter of liposomes), lipid composition (homogeneous and phase-separated liposomes) and/or peptide density to identify inhibitors that have optimal activities in toxin-challenge and spore-challenge animal models.
The second aim of this proposal is to perform preformulation studies to optimize inhibitor solubility and stability. Inhibitor solubility will be assessed in buffers made within a clinically suitable range of pH values, and in the presence of co- solvents and surfactants. Accelerated, intermediate and long term stability tests will be used to identify formulations most suitable for stockpiling of the inhibitor.
The third aim of this proposal is to determine the pharmacokinetics, tissue distribution, mass balance and toxicity of inhibitors. These data will assess the suitability of an inhibitor for clinical use and will be used to support an Investigational New Drug application. Relevance: The goal of this research is to develop a drug that blocks anthrax toxin. An anti-toxin therapeutic will be an important addition to the antibiotic therapy used to treat patients with anthrax. ? ? ?
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