Development and testing of polyvalent anthrax toxin inhibitors

多价炭疽毒素抑制剂的开发和测试

• 批准号: 7936885
• 负责人: JEREMY S MOGRIDGE
• 金额: $ 117.5万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936885
• 关键词: Animal Model; Animals; Anthrax disease; Antibiotic Therapy; Antigens; Bacillus anthracis; Bacillus anthracis spore; Binding; Buffers; Caliber; Cause of Death; Cells; Cessation of life; Cleaved cell; Clinical; Clinical Trials; Cold Chains; Complex; Data; Development; Disease Progression; Drug Formulations; Drug Kinetics; Drug or chemical Tissue Distribution; Edema; Endocytosis; Equilibrium; Exhibits; Funding; Glutamic Acid; Goals; In Vitro; Intoxication; Investigational New Drug Application; Lipids; Liposomes; Mammalian Cell; Marketing; Molecular Weight; Mus; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phase; Poly-5; Polymers; Process; Property; Proteins; Rattus; Reproduction spores; Research; Solubility; Solvents; Symptoms; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; Toxin; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; antigen binding; base; copolymer; density; edema factor; in vivo; inhibitor/antagonist; prevent; receptor; scaffold; stability testing; surfactant; therapeutic target

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.

描述（由申请人提供）：拟议工作的目的是开发一种炭疽毒素抑制剂，该抑制剂在体内有效，适用于临床使用。炭疽毒素是由炭疽杆菌分别分泌的三种蛋白质的组合，并形成由哺乳动物细胞内化的毒性复合物。保护性抗原（PA）结合细胞受体并被弗林蛋白酶样蛋白酶切割，这导致细胞相关的PA 63片段的七聚化。七聚化允许酶毒素组分、水肿因子和致死因子的结合，并触发这些复合物的内吞作用和细胞的中毒。由于炭疽毒素是一种重要的毒力因子，并导致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是一个理想的靶点，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，这两种毒素都诱导组织损伤并导致死亡。我们已经附加了多个拷贝的PA 6s七聚体结合抑制肽的聚-L-谷氨酸（PLGA）和脂质体支架，以产生多价分子，这是几个数量级更有效的单体肽在体外和中和炭疽毒素在体内。本提案中描述的多价抑制剂的优点是：1）它们阻断两种毒素; 2）它们是从常规临床使用的无毒支架合成的（脂质体）或已在临床试验中显示为安全的（PLGA）; 3）合成方法容易且可规模化; 4）多价提供效力的显著增强;和5）基于PLGA的抑制剂不需要冷链。该提案的第一个目的是优化多价炭疽毒素抑制剂的体内功效。我们将合成有限数量的基于PLGA和基于脂质体的抑制剂，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（均质和相分离的脂质体）和/或肽密度方面不同，以鉴定在毒素攻击和孢子攻击动物模型中具有最佳活性的抑制剂。该提案的第二个目的是进行预配制研究，以优化抑制剂的溶解度和稳定性。将在临床适用的pH值范围内制备的缓冲液中以及在存在共溶剂和表面活性剂的情况下评估抑制剂溶解度。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制剂。本提案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用于临床的适用性，并将用于支持研究性新药申请。相关性：这项研究的目标是开发一种阻止炭疽毒素的药物。抗毒素治疗将是用于治疗炭疽病患者的抗生素治疗的重要补充。