Synthesis of Metal-Free Magnetic Resonance Imaging Contrast Agents

描述（由申请人提供）：本项目的总体目标是开发生物稳定的有机自由基作为磁共振成像（MRI）的造影剂（CA）。这种不含金属的有机CA将在对比增强MRI方面取得突破，同时避免与有毒金属离子相关的风险。虽然这些药物将特别有利于肾功能受损的患者，这些患者在给予顺磁性钆螯合物（GBCA）进行MRI程序后发生肾源性系统性纤维化（NSF）的风险增加，但新药物也可能使一般人群受益。所提出的工作的目的是合成有机自由基造影剂（ORCAs），提供高质量的体内MR图像。在开发用于MRI的实用ORCA中的长期存在的障碍是设计和合成具有足够长的体内寿命、高1H水弛豫率（r1）和高水弛豫率（r2）的中等分子大小的顺磁性有机化合物。 溶解度我们提出了一种设计策略，从氮氧自由基，是高度耐还原和利用有关树枝状聚合物和聚乙二醇化，特别是其应用于药物设计的先验知识，以实现具有优化的MRI性能的代理商。拟议的项目有三个具体目标。具体目标1：优化ORCA的1H水弛豫率r1（在3特斯拉下）和亲水性，以实现分子r1 = 10 mM，至少是临床CA的两倍（分子r1 = 5 mM λ）。 ORCA衍生自通过长线性和/或支链PEG链PEG化与第4代聚丙烯亚胺（PPI-G4）树枝状聚合物缀合的氮氧化物。拟定的工作将为具体目标2提供基础：优化分子r1 ≥ 10 mM的ORCA的分子大小，以获得半径1 nm ≤ rSE ≤ 2 nm的药物，这些药物将进行有效的肾过滤/排泄。将测定ORCA的体外毒性和水弛豫率，以及影响分子r1的性质，如旋转相关时间和电子自旋弛豫时间，以确定四种最佳ORCA。具体目标3：评价ORCA用于体内MRI的有效性。将在3 T的临床视野下通过正常小鼠的MRI和荷瘤异种移植物小鼠的动态对比增强（DCE）MRI确定4种选定ORCA的最低有效剂量、时间过程和肿瘤血管通透性（转移常数Ktranss）。将通过组织学检查评价ORCA的初步毒性。将选择最有效的ORCA，用荧光探针标记，然后通过电子顺磁共振和荧光光谱进行离体生物分布研究。这些研究将确定ORCA进一步开发用于人类的适用性。基于生物稳定有机自由基的无金属MRI造影剂的开发将拓展生物医学成像的前沿。有机合成的模块化使得能够进行有效的结构修饰以调整特定的性质（氧化还原、溶解度和毒性）。这种基于有机物的试剂将改善疾病的早期检测和诊断。

DESCRIPTION (provided by applicant): The overall goal of this project is the development of biostable organic radicals as contrast agent (CA) for magnetic resonance imaging (MRI). Such metal-free, organic-based CAs will provide a breakthrough in contrast-enhanced MRI while avoiding the risk associated with toxic metal ions. While the agents would especially benefit patients with impaired kidney function, who are at increased risk of developing nephrogenic systemic fibrosis (NSF) following administration of paramagnetic gadolinium chelates (GBCAs) for MRI procedures, the new agents may also benefit the general population. The objective of the proposed work is to synthesize organic radical contrast agents (ORCAs) that provide high quality in vivo MR images. The long-standing obstacle in the development of a practical ORCA for MRI is the design and synthesis of paramagnetic organic compounds of moderate molecular size that possess sufficiently long in vivo lifetime, high 1H water relaxivity (r1), and high water solubility. We propose a design strategy to prepare ORCAs from nitroxide radicals that are highly resistant to reduction and exploit prior knowledge about dendrimers and PEGylation, particularly their applications to drug design, to achieve agents with optimized MRI properties. The proposed project has three specific aims. Specific aim 1: optimize 1H water relaxivity r1 (at 3 Tesla) and hydrophilicity of ORCAs to achieve molecular r1 = 10 mMs, at least twice that of the clinical CAs (molecular r1 ≈ 5 mM‾¹s‾¹). The ORCAs are derived from nitroxides conjuated to a generation 4 polypropylenimine (PPI-G4) dendrimer through PEGylation with long linear and/or branched PEG chains. The proposed work will provide a foundation for specific aim 2: optimize molecular size of the ORCAs with molecular r1 ≥ 10 mM‾¹s‾¹ to achieve agents with th radii 1 nm ≤ rSE ≤ 2 nm that would undergo efficient renal filtration/excretion. In vitro toxicty and water relaxivity of the ORCAs, as well as the properties that affect molecular r1, such as rotational correlation time and electron spin relaxation time, will be determined, to identify the four best ORCAs. Specific aim 3: evaluate effectiveness of ORCAs for in vivo MRI. Lowest effective dose, time course, and tumor vascular permeability (transfer constant Ktrans) for the four selected ORCAs will be determined at the clinical field of 3 Tesla by MRI of normal mice and by dynamic contrast enhanced (DCE) MRI of mice bearing tumor xenografts. Preliminary toxicity of the ORCA will be evaluated by histological examination. The most effective ORCA will be selected, labeled with a fluorescence probe, and then subjected to ex vivo biodistribution studies by electron paramagnetic resonance and fluorescence spectroscopy. These studies will determine suitability of ORCA for further development towards human use. Development of metal-free MRI contrast agents based on biostable organic radicals will expand the frontier of biomedical imaging. Modularity of organic synthesis enables efficient structural modifications for specific property tuning (redox, solubility, and toxicity). Such organic-based agents will improve early detection and diagnosis of diseases.