Development and testing of polyvalent anthrax toxin inhibitors

描述（由申请人提供）：拟议工作的目标是开发一种体内有效且适合临床使用的炭疽毒素抑制剂。炭疽毒素是由炭疽芽孢杆菌分别分泌的三种蛋白质的组合，形成有毒复合物，被哺乳动物细胞内化。保护性抗原（PA）结合细胞受体，并被类似呋喃蛋白的蛋白酶裂解，导致细胞相关的PA63片段七聚化。七聚化允许结合酶毒素成分、水肿因子和致死因子，并触发这些复合物的内吞作用和细胞中毒。由于炭疽毒素是一种重要的毒力因子，并负责炭疽的主要症状和死亡，因此该毒素是治疗干预的主要目标。特别是PA是一个理想的靶标，因为它是炭疽毒素、水肿毒素和致死毒素这两种毒素的共同成分，这两种毒素都能引起组织损伤并导致死亡。我们已经将PA6s七聚体结合抑制肽的多个拷贝附着在聚l -谷氨酸（PLGA）和脂质体支架上，以产生多价分子，这些多价分子在体外比单体肽强几个数量级，并在体内中和炭疽毒素。本文所述的多价抑制剂的优点是：1)它们可以阻断两种毒素；2)它们是由临床常规使用的无毒支架（脂质体）或在临床试验中被证明是安全的（PLGA）合成的；3)合成工艺简便、可扩展；4)多价性能显著增强效力；5)基于plga的抑制剂不需要冷链。本研究的第一个目的是优化多价炭疽毒素抑制剂的体内疗效。我们将合成有限数量的基于PLGA和脂质体的抑制剂，这些抑制剂在体外表现出高效力，并且在大小（PLGA的分子量；脂质体的直径）、脂质组成（均质和相分离脂质体）和/或肽密度上有所不同，以确定在毒素挑战和孢子挑战动物模型中具有最佳活性的抑制剂。本提案的第二个目的是进行配方前研究，以优化抑制剂的溶解度和稳定性。抑制剂的溶解度将在临床适宜的pH值范围内，在共溶剂和表面活性剂的存在下进行评估。加速、中期和长期稳定性试验将用于确定最适合储存抑制剂的配方。本建议的第三个目的是确定抑制剂的药代动力学、组织分布、质量平衡和毒性。这些数据将评估抑制剂临床使用的适用性，并将用于支持研究性新药申请。相关性：本研究的目的是开发一种阻断炭疽毒素的药物。抗毒素治疗将是用于治疗炭疽患者的抗生素治疗的重要补充。

DESCRIPTION (provided by applicant): 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.