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）的硼中子捕获治疗（BNCT）中，迫切需要新的和更有效的硼递送剂。一类硼化的非天然环状氨基酸在动物模型中显示出对肿瘤的显著选择性，这远远优于目前临床BNCT中使用的药剂上级。这些氨基酸之一的硼类似物，1-氨基-3-硼环戊烷羧酸，显示肿瘤与血液的比率为8，肿瘤与正常脑的比率接近21。该化合物在硼的肿瘤靶向方面也显示出顺式和反式异构体的外消旋混合物之间的显著差异。这意味着该化合物的L-和D-形式的进一步分离可以潜在地将肿瘤靶向增强到甚至比异构体混合物所提供的更高的程度。由于所有这些测量都是在均质肿瘤和正常组织中进行的，因此目前缺乏对硼的亚细胞位置和异构体之间差异的基本了解。一个专家小组聚集在目前的建议，了解亚细胞交付和细胞周期偏好的10个硼化非天然氨基酸（三个母分子和所有的异构体）在T98 G人胶质母细胞瘤和F-98大鼠胶质瘤细胞系与二次离子质谱（西姆斯）的离子显微镜技术。西姆斯能够以500 nm的空间分辨率对细胞和组织中的元素（同位素）梯度进行定量成像，并且已经在BNCT中发展到非常精确的程度，用于低温制备细胞中的硼成像。将使用西姆斯在亚细胞分辨率下评价硼化非天然氨基酸及其异构体的硼递送特性。从3种母体分子及其异构体（共10种化合物）的这些体外研究中，我们将每年从每个母体分子中选择一种硼化的非天然氨基酸。将在GBM的F98大鼠神经胶质瘤模型中测试该选定分子选择性递送硼原子至正常脑中的浸润肿瘤细胞的能力。低温制备的组织中的西姆斯分析将提供主要肿瘤块、正常脑和正常脑中的浸润肿瘤细胞的硼靶向信息。西姆斯是目前唯一能够直接测量浸润性肿瘤细胞中硼的技术。由于正常脑中的浸润性肿瘤细胞是临床BNCT的主要目标，这些独特的西姆斯观察将提供与GBM高度相关的关于硼化非天然氨基酸的硼递送特征的最引人注目的观察。在拟议拨款的第三年年底，将选出最有前途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，拟议的亚细胞西姆斯研究异构形式的硼化非天然氨基酸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，脑癌，多形性胶质母细胞瘤（GBM）没有可行的治疗方法。硼中子俘获疗法（BNCT）正在开发中，用于治疗GBM。西姆斯离子显微镜的亚细胞/单细胞同位素成像技术将用于理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递送硼的机制方面。西姆斯是目前唯一可靠地测量硼原子在浸润性胶质母细胞瘤细胞和正常大脑之间分配的技术。这些都是BNCT成功的重要观察结果。

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.