Subcellular/Cell Cycle Boron-Delivery Studies of Unnatural Amino Acids by SIMS

描述（由申请人提供）：在多形性胶质母细胞瘤（GBM）的硼中子俘获治疗（BNCT）中，迫切需要新的、更有效的硼递送剂。一类硼化的非天然环氨基酸在动物模型中显示出对肿瘤的显著选择性，这远远优于目前在临床BNCT中使用的药物。其中一种氨基酸的硼类似物，1-氨基-3-硼环戊烷羧酸，显示出肿瘤与血液的比率为8，肿瘤与正常脑的比率接近21。该化合物还显示出顺式和反式异构体的外消旋混合物在硼靶向肿瘤中的显着差异。这意味着进一步分离该化合物的L-和D-形式可能会比同分异构体混合物提供更高程度的肿瘤靶向性。由于所有这些测量都是在均匀化的肿瘤和正常组织中进行的，因此目前对硼的亚细胞位置和异构体之间的差异缺乏基本的了解。一组专家在目前的建议中集合，利用基于离子显微镜的二次离子质谱（SIMS）技术，了解T98G人胶质母细胞瘤和F-98大鼠胶质瘤细胞系中10种硼化非天然氨基酸（3种亲本分子及其所有异构体）的亚细胞递送和细胞周期偏好。SIMS能够在500 nm的空间分辨率下对细胞和组织中的元素（同位素）梯度进行定量成像，并且在BNCT中对低温制备的细胞进行硼成像已经发展到非常高的精度。硼化非天然氨基酸及其异构体将用SIMS在亚细胞分辨率下评估其硼传递特性。从这些3个亲本分子及其异构体（共10种化合物）的体外研究中，我们将每年从每个亲本分子中选择一个硼化非天然氨基酸。该分子将在F98大鼠GBM胶质瘤模型中测试其选择性递送硼原子到正常脑内浸润肿瘤细胞的能力。低温制备组织的SIMS分析将提供主要肿瘤肿块、正常大脑和正常大脑浸润性肿瘤细胞的硼靶向信息。SIMS是目前唯一能够直接测量浸润性肿瘤细胞中硼含量的技术。由于正常脑内浸润的肿瘤细胞是临床BNCT的主要目标，这些独特的SIMS观察结果将提供与GBM高度相关的硼化非天然氨基酸的硼传递特性的最令人信服的观察结果。在计划拨款的第三年结束时，将选择一种最有希望的候选药物进行临床BNCT测试。从基础化学生物学的角度来看，以及BNCT，拟议的亚细胞模拟模拟研究硼化非天然氨基酸的异构体形式提供了一个独特的机会，产生宝贵的新信息。公共卫生相关性：目前，没有可行的治疗脑癌多形性胶质母细胞瘤（GBM）的方法。硼中子俘获疗法（BNCT）是治疗GBM的一种新方法。SIMS离子显微镜的亚细胞/单细胞同位素成像技术将用于了解硼化非天然氨基酸及其异构体在细胞培养和GBM动物模型中向肿瘤细胞输送硼的机制。SIMS是目前唯一可靠地测量浸润性胶质母细胞瘤细胞和正常大脑之间硼原子分配的技术。这些是BNCT成功的基本观察结果。

DESCRIPTION (provided by applicant): There is a pressing need for new and more efficient boron delivery agents to tumor cells in Boron Neutron Capture Therapy (BNCT) of glioblastoma multiforme (GBM). A class of boronated unnatural cyclic amino acids has shown a remarkable selectivity to tumors in animal models, which is far superior to currently used agents in clinical BNCT. The boron analogue of one of these amino acids, 1-amino-3- boronocyclopentanecarboxylic acid, has shown a tumor to blood ratio of 8 and a tumor to normal brain ratio of nearly 21. This compound has also shown a remarkable difference between racemic mixtures of cis- and trans- isomers in tumor targeting of boron. This implies that further separation of the L- and D- forms of this compound may potentially enhance tumor targeting to an even higher degree than that provided by the isomeric mixture. Since all of these measurements were made in homogenized tumor and normal tissues, the basic understanding of the subcellular location of boron and differences between the isomers is currently missing. A team of experts is assembled in the current proposal for understanding subcellular delivery and cell cycle preferences of 10 boronated unnatural amino acids (three parent molecules and all of their isomers) in T98G human glioblastoma and F-98 rat glioma cell lines with the secondary ion mass spectrometry (SIMS) based technique of ion microscopy. SIMS is capable of quantitatively imaging elemental (isotopic) gradients in cells and tissues at 500 nm spatial resolution and has been developed to a remarkable precision in BNCT for boron imaging in cryogenically prepared cells. The boronated unnatural amino acids and their isomers will be evaluated for their boron delivery characteristics at subcellular resolution with SIMS. From these in vitro studies of 3 parent molecules and their isomers (a total of 10 compounds), we will select each year one boronated unnaturated amino acid from each parent molecule. This selected molecule will be tested in F98 rat glioma model of GBM for its ability to selectively deliver boron atoms to infiltrating tumor cells in the normal brain. SIMS analysis in cryogenically prepared tissues will provide the boron- targeting information of the main tumor mass, the normal brain, and the infiltrating tumor cells in the normal brain. SIMS is currently the only technique capable of direct measurements of boron in infiltrating tumor cells. Since infiltrating tumor cells in the normal brain are the main targets of clinical BNCT, these unique SIMS observations will provide the most compelling observations highly relevant to GBM on the boron-delivery characteristics of boronated unnatural amino acids. By the end of the third year of the proposed grant, a most promising candidate drug will be selected for testing in clinical BNCT. From the point of view of fundamental chemical biology, as well as BNCT, the proposed subcellular SIMS studies of isomeric forms of boronated unnatural amino acids offer a unique opportunity to produce invaluable new information. PUBLIC HEALTH RELEVANCE: Currently, there is no viable therapy for brain cancer, glioblastoma multiforme (GBM). The boron neutron capture therapy (BNCT) is under development for the treatment of GBM. The subcellular/single cell isotopic imaging technique of SIMS ion microscopy will be used for understanding mechanistic aspects of boronated unnatural amino acids and their isomers in delivering boron to tumor cells in cell culture and animal models of GBM. SIMS is currently the only technique for reliably measuring the partitioning of boron atoms between the infiltrating glioblastoma cells and the normal brain. These are essential observations for the success of BNCT.