Metal Chelate Conjugated Dendrimer Constructs for Diagnosis and Therapy

基于树状大分子的磁共振造影剂消除了血清白蛋白或类似大小的线性聚合物MRI造影剂的许多缺陷。这是由于它们被创造出来的迭代聚合合成，然后促进了树突分子的控制大小和形状，同时提供了可重复化学的手段，这是这些药物临床翻译的关键。为了制造具有树状大分子的MRI造影剂，我们在实验室开发了用螯合Gd（III）修饰的树状大分子的末端伯胺。这些试剂的摩尔弛豫率是目前批准的MRI造影剂Magnevist的6倍。利用PAMAM和聚丙烯亚胺或DAB树突状物获得了出色的常规全身磁共振成像和3D T-O-F磁共振血管成像。过去的研究结果表明，大分子螯合物共轭树突状聚合物基Gd(III) MR造影剂可以通过调整基本标准来适应各种应用：生成（分子量大小）、核心元素（亲脂性和电荷）、PEG偶联、赖氨酸共给药（肾脏清除）和偶联靶向载体（分子靶向）。基于PAMAM的药物可以在200微米尺度上精确成像小鼠肿瘤血管系统。基于DAB的试剂具有选择性，其中0.3 mm转移性肝肿瘤的反向对比图像被检测到。这些基于树突状分子的药物也被选择性靶向，不仅通过与抗体结合，而且通过其他载体，如亲和素，将异常高水平的Gd（III）递送到弥散性腹腔内卵巢癌肿瘤中。这项研究与光学显像剂一起进行，与我们创建的多模态树状聚合物显像剂并行。在MRI试剂中加入近红外光学成像染料，以提高灵敏度水平，以补充MRI成像的分辨率，为淋巴和前哨淋巴结的成像提供了额外的灵敏度水平，可以设想转化为术中场景，其中MRI成像和绘图将补充实时手术干预和恶性肿瘤切除。虽然化学方法建立了制造这种大分子试剂的能力，但成像结果导致靶向性受损，这决定了这些试剂需要非常仔细的系统研究，并结合同样仔细定义的特性。最后，由于需要重新发明这一领域，将其从水化学移回有机相溶剂，以提高表征和产率的一致性，促使了Gd（III）配合物与树状大分子缀合的新螯合化学。这种化学也发展出了对已建立的双功能螯合剂的专门类似物的需求，以解决主动靶向树突基显像剂所需的位点特异性偶联化学的发展。与此同时，最近不太稳定的MRI造影剂对NFS相关Gd（III）毒性的影响，促使所有这些研究的核心在双功能螯合剂的选择上应用新的方向性的项目完全停止。因此，所有正在进行的项目都是使用1B4M-DTPA双功能螯合剂完成的，而所有新项目都被搁置，直到通过该科自身的综合努力获得足够数量的双功能DOTA，而不是购买这种剂。虽然这项工作在过去一年中已经到位，但所有这些项目现在都转向使用DOTA对Gd（III）偶联策略进行预络合，以消除旨在简单地将这些药物转化为临床使用的表征复杂性。验证这一转变的研究结果表明，不仅可以采用这种策略，而且可以通过这种方法获得更大的摩尔弛豫。树突状分子基药剂相对于低分子量药剂的优越优势继续得到非常清楚的证明。在开发基于树突状分子的药物的同时，与NINDS研究人员长期合作开发化疗药物CED的替代标记物继续向前推进，包括所有上述化学修饰，以尽可能安全的形式将这项技术推进到临床。一项涉及这项技术的美国专利于今年发布，并引起了相当大的关注和兴趣，这将有助于将这项技术转化为临床。不幸的是，与分子成像计划合作的MRI和其他成像模式剂的研究由于缺乏合作和对驻留在其中的仪器的访问而被有效终止，这些仪器本应是所有NCI研究人员的资源。然而，与放射学、CC、PER部门、CC和NIMH以及校外研究人员的合作已经取代了NCI的分子成像项目。

Macromolecular MRI contrast agents based upon dendrimers obviate many of the deficiencies of serum albumin or linear polymer based MRI contrast agents of comparable size. This is due to the iterative polymeric synthesis by which they are created that then promotes a controlled size and shape of the dendrimer that concomitantly then provides the means for reproducible chemistry that is key to the clinical translation of such agents. To create MRI contrast agents with dendrimers, the terminal primary amines of dendrimers modified with chelated Gd(III) are developed in our laboratories. These reagents possess a molar relaxivity 6 times that of Magnevist, the currently approved MRI contrast agent. Excellent conventional whole body MR imaging and 3D T-O-F MR angiograms have been obtained with PAMAM and polypropyleneimine or DAB dendrimer based agents. Past results have established that macromolecular chelate conjugated dendrimer based Gd(III) MR contrast agents can be tuned for various applications by adjusting fundamental criteria: generation (MW & size), core elements (lipophilicity & charge), PEG conjugation, lysine co-administration (renal clearance), and conjugation to targeting vectors (molecular targeting). PAMAM based agents have imaged murine tumor vasculature accurately at the 200 micron scale. DAB based agents have selective properties wherein reverse contrast images of 0.3 mm metastatic liver tumors were detected. These dendrimer based agents have also been selectively targeted, not only by conjugation to antibodies, but by other vectors, such as avidin to deliver exceptionally high levels of Gd(III) into disseminated intraperitoneal ovarian cancer tumor. This study done in conjunction with an optical imaging agent runs in parallel with our creation of multi-modality dendrimer based imaging agents. The incorporation of a NIR optical imaging dye into the MRI agent to add an enhanced level of sensitivity to complement the resolution of the MRI imaging provided an additional level of sensitivity for the imaging of lymphatics and sentinel nodes that can be envisioned as being translated to an intraoperative scenario wherein MRI imaging and mapping would supplement real-time surgical intervention and excision of malignancy. While the chemistry established the ability to create such macromolecular agents, the imaging resulted in compromised targeting which defined that these agents require very careful systematic investigation combined with equally carefully defined characterization. Lastly, new chelation chemistry for conjugation of Gd(III) complexes to dendrimer has been prompted by the need to re-invent this filed moving it from aqueous chemistry back to organic phase solvents to enhance both characterization and consistency of yields. This chemistry has also evolved out of the need for specialized analogs of established bifunctional chelation agents to address the development of site-specific conjugation chemistry required for actively targeted dendrimer based imaging agents. In parallel to this effort, the very recent impact on NFS related Gd(III) toxicity of less than adequately stable MRI contrast agents prompted a complete halting of projects with an application of new directionality in the choice of bifunctional chelating agent at the heart of all of these studies. Thus, all ongoing projects were completed using the 1B4M-DTPA bifunctional chelate while all new projects were put on hold until adequate amounts of bifunctional DOTA became available through the synthesis efforts of the Section itself as opposed to purchase of this agent. While this effort was put into place over the past year, all of these projects have now moved over to use of a pre-complexation of the Gd(III) conjugation strategy using DOTA to eliminate a characterization complexity intended to simply translation of these agents into clinical use. Results from the studies to validate this transformation have revealed that not only can such a strategy be employed, but that far greater molar relaxivity can be achieved by this means. The exquisite advantages of the dendrimer based agents over low molecular weight agents continue to be very clearly demonstrated. In parallel to the development of dendrimer based agents, a long-term collaboration with NINDS investigators to develop a surrogate marker for CED of chemotherapeutic drugs continues to move forward with all of the above noted chemical modifications included to advance this technology into the clinic in the safest format possible. A US patent covering this technology was issued this year and is attracting considerable attention and interest that should contribute to translation of this technology into the clinic. Studies of MRI and other imaging modality agents in collaboration with the Molecular Imaging Program have unfortunately been effectively terminated due to a lack of cooperation and access to instrumentation residing therein what was to be a resource for all NCI researchers. However, collaboration with Radiology, CC, the PER Dept, CC, and NIMH , and extramural researchers have replaced the Molecular Imaging Program, NCI.