Mechanism-based Optimization of Tirapazamine Analogs

这个项目有三个广泛的目标，每个目标都与这个项目的总体目标有关，即开发肿瘤缺氧细胞的新疗法。跨越所有三个目标的进一步整合主题是使用多细胞层(MCL)培养来量化药物及其活性代谢物的血管外运输。第一个目标是开发一种改进的低氧选择性细胞毒素替拉帕明(TPZ)的类似物，目前该毒素正处于第三阶段临床试验。该方法是基于我们的TPZ空间分辨药代动力学/药效学(PK/PD)模型的见解，该模型明确考虑了血管外转运。该模型表明，通过增加血管外扩散系数、优化新陈代谢速率和改善导致细胞毒性的代谢部分(Lambda)，可以获得较大的活性收益。我们将使用PK/PD模型通过筛选来指导化合物的进展，并将评估模型中每个组件的药物构效关系(例如组织中的扩散系数)。我们的方法将检验以下特定假设：(I)DNA靶向可用于提高细胞毒力和lambda，以及(Ii)增加TPZ自由基的细胞内扩散范围是增加lambda的进一步策略。第二个目标是表征由重组梭状芽孢杆菌表达的酶激活前药物在肿瘤坏死和缺氧区产生的旁观者效应。这将涉及鉴定由大肠杆菌硝基还原酶、β-葡萄糖醛酸酶和β-半乳糖苷酶激活的前体药物所产生的活性代谢物，并量化活性物质在MCL中的血管外运输和由此产生的旁观者杀伤。第三个目标是确定从筛选的针对HIF-1α上调的新药中筛选出的哪些初始“热门”药物具有足够好的血管外运输特性，以保证进一步的开发。

This Project has three broad goals, each of which relate to the overall objective of this Program to develop novel therapies for hypoxic cells in tumors. A further integrating theme across all three goals is use of multicellular layer (MCL) cultures to quantify extravascular transport of drugs and their active metabolites. The first goal is to develop an improved analog of the hypoxia-selective cytotoxin tirapazamine (TPZ), which is currently in phase III clinical trial. The approach is based on insights from our spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) model for TPZ, which explicitly takes extravascular transport into account. This model demonstrates that large gains in activity could be achieved by increasing extravascular diffusion coefficients, optimizing rates of metabolism, and improving the fraction of metabolism that contributes to cytotoxicity (lambda). We will use the PK/PD model to guide advancement of compounds through screening, and will evaluate drug structure-activity relationships for each component of the model (e.g. diffusion coefficient in tissue). Our approach will test the specific hypotheses that (i) DNA targeting can be used to improve the cytotoxic potency and lambda, and (ii) increasing the intracellular diffusion range of the TPZ radical is a further strategy for increasing lambda. The second goal is to characterize bystander effects resulting from activation of prodrugs by enzymes expressed by recombinant clostridia in necrotic and hypoxic regions of tumors. This will involve the identification of active metabolites resulting from activation of prodrugs by E. coli nitroreductase, beta-glucuronidase and beta-galactosidase, and to quantify extravascular transport of the active species, and the resulting bystander killing, in MCLs. The third goal is to identify which of the initial "hits" from a screen for novel drugs targeting HIF-1alpha upregulation have good enough extravascular transport properties to warrant further development.