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

描述(申请人提供)：在多形性胶质母细胞瘤(GBM)的硼中子俘获疗法(BNCT)中，迫切需要新的和更有效的硼输送到肿瘤细胞。一类含硼的非天然环状氨基酸在动物模型中对肿瘤具有显著的选择性，远远优于目前临床上使用的BNCT药物。其中一种氨基酸1-氨基-3-硼环戊烷羧酸的硼类似物显示，肿瘤与血液的比率为8，肿瘤与正常大脑的比率接近21。这种化合物在肿瘤靶向硼的顺式和反式异构体混合物中也显示出显著的差异。这意味着进一步分离这种化合物的L和D-型可能会潜在地将肿瘤靶向性提高到比异构体混合物提供的更高的程度。由于所有这些测量都是在匀浆的肿瘤和正常组织中进行的，因此目前还缺乏对硼的亚细胞位置和异构体之间的差异的基本了解。为了解10种含硼非天然氨基酸(三种亲本分子及其所有异构体)在T98G人胶质母细胞瘤和F-98大鼠胶质瘤细胞系中的亚细胞递送和细胞周期偏好，利用基于二次离子质谱仪(SIMS)的离子显微镜技术，组建了一个专家小组。SIMS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，并已在BNCT中用于低温制备的细胞中的硼成像，具有显著的精度。含硼的非天然氨基酸及其异构体将用SIMS在亚细胞分辨率下评估它们的硼输送特性。从这些对3个母体分子及其异构体(总共10个化合物)的体外研究中，我们将每年从每个母体分子中选择一个含硼的非天然氨基酸。这个选定的分子将在F98大鼠胶质瘤模型GBM中进行测试，以测试其选择性地将硼原子输送到正常大脑中浸润性肿瘤细胞的能力。低温制备的组织中的SIMS分析将提供主要肿瘤肿块、正常脑以及正常脑组织中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的技术。由于正常大脑中浸润性肿瘤细胞是临床BNCT的主要靶点，这些独特的SIMS观察将提供与GBM高度相关的最令人信服的观察结果，以了解含硼非天然氨基酸的硼输送特性。到拟议拨款的第三年结束时，将选出一种最有希望的候选药物在临床上进行BNCT测试。从基础化学生物学和BNCT的角度来看，拟议的亚细胞SIMS研究硼化非天然氨基酸的异构体提供了一个独特的机会来产生宝贵的新信息。公共卫生相关性：目前，还没有可行的治疗脑癌的方法，即多形性胶质母细胞瘤(GBM)。硼中子俘获疗法(BNCT)正在开发中，用于治疗GBM。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。这些都是BNCT成功的基本观察。

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.