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的迭代合成，促进了树突分子的大小和形状的控制，同时产生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，我们利用实验室开发的螯合Gd（III）技术对树状大分子的末端伯胺进行修饰。历史上，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。大分子螯合物偶联树突状分子Gd(III) MR造影剂可以通过调整基本标准来调整各种应用：生成（分子量和大小），核心元素（亲脂性和电荷），PEG偶联（延长循环并减少肝脏和其他器官的摄取），赖氨酸共给药（肾脏清除），以及偶联靶向载体（分子靶向）。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。这是与光学显像剂一起完成的，同时我们也创造了多模态树突基显像剂。在MRI试剂中加入近红外光学成像染料，增加了灵敏度水平，以补充MRI成像的分辨率，并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，并结合同样仔细的定义特性。由于需要重新发明这一领域，将其从水化学转移到有机相溶剂，以提高表征和产量的一致性，促使了Gd（III）配合物与树状大分子缀合的新螯合化学。这种化学还发展出了已建立的双功能螯合剂的专门类似物（专利申请），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，例如，针对独特硫醇残基的马来酰亚胺，或用于点击化学偶联策略的炔或叠氮基团功能化的试剂。由于使用不够稳定的MRI造影剂和低肾排泄引起的NFS相关Gd（III）毒性的影响，在双功能螯合剂的选择上应用新的方向性的项目已经完全停止。然而，重定向使用DOTA作为双功能螯合剂，在与所有靶向载体结合之前预先形成Gd（III）配合物，已被证明可以成功地解决毒性问题。这项工作是在所有MR对比项目完全迁移到使用DOTA的Gd（III）共轭物的预络合策略的情况下进行的，该策略还消除了由于创建极其困难的产品混合物而导致的表征复杂性，这些混合物限制了可重复性，从而使这些药物的临床翻译变得复杂。结果证实，这种转变不仅是一种成功的策略，尽管有溶解性降低的警告（不是真的），而且提供了更大的摩尔弛豫。我们报告了5倍于先前技术的增强，同时将Gd（III）共轭到树状聚合物上的实际物理量减少了65%，进一步提高了安全边际。这一结果的影响遍及所有的大分子磁共振造影剂，而不考虑平台，以充分解决安全性、特性和可重复性问题，从而进一步提高了这类药物临床翻译的整个领域的潜力。树突状分子基药剂优于低分子量药剂（如Magnevist）的优势，继续得到非常清楚的证明。与此同时，这种改进消除了毒性问题，从而产生了优越的基于树突状物的药物，通过将半胺核心树突状物衍生的树突以1:1的形式与抗体片段偶联，最终实现了主动靶向树突状物造影剂的能力。虽然取得了成功，但分离和纯化都是具有挑战性的，因为所采用的树状分子世代以及相对较低的松弛性结果表明需要继续向更高的树状分子世代移动。该研究继续与PEG附加三功能显像剂的创建同时进行，该显像剂将允许将放射探针（PET成像）与光学探针结合。专利申请继续被起诉，同时出版物向该领域揭示了这项技术，这应该证明它对卫生与公众服务部更有价值，也应该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核磁共振成像和其他成像模式剂的研究已被终止，因为缺乏合作和访问驻留在其中的仪器，这些仪器本应是所有NCI研究人员的资源，尽管有相反的协议。然而，与放射学，CC， PET部门，CC， NIMH和约翰霍普金斯大学的校外研究人员建立的合作继续取得成果。

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 synthesis by which they are created that promotes controlled size and shape of the dendrimer concomitantly generating the means for reproducible chemistry key to the clinical translation of such agents. To create MRI contrast agents with dendrimers, the terminal primary amines of dendrimers are modified with chelated Gd(III) technology developed in our laboratories. Historically, these reagents demonstrated 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 using PAMAM and polypropyleneimine dendrimer-based agents. 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 (prolong circulation and minimize liver & other organ uptake), lysine co-administration (renal clearance), and conjugation to targeting vectors (molecular targeting). 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 was done in conjunction with an optical imaging agent in parallel with our creation of multi-modality dendrimer based imaging agents. The incorporation of a NIR optical imaging dye into the MRI agent added an enhanced level of sensitivity to complement the resolution of the MRI imaging and 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 careful defined characterization. New chelation chemistry for conjugation of Gd(III) complexes to dendrimer has been prompted by the need to re-invent this field moving it from aqueous chemistry to organic phase solvents to enhance both characterization and consistency of yields. This chemistry has also evolved specialized analogs of established bifunctional chelation agents (patent filed) to address development of site-specific conjugation chemistry required for actively targeted dendrimer based imaging agents e.g, maleimides targeting a unique thiol residue, or agents functionalized with alkyne or azide groups for click chemistry conjugation strategies. The impact of NFS related Gd(III) toxicity resultant from use of less than adequately stable MRI contrast agents combined with low renal excretion had prompted a complete halt of projects with application of new directionality in the choice of bifunctional chelating agent. Redirection towards the use of DOTA as the bifunctional chelating agent, however, with the Gd(III) complex pre-formed prior to conjugation to all targeting vectors has been demonstrated to successfully traverse toxicity concerns. This effort was put into place with all of the MR contrast projects fully migrated to the exclusive use of a pre-complexation of the Gd(III) conjugate strategy using DOTA to also eliminate characterization complexity resulting from the creation of exceedingly difficult to characterize mixtures of products that limited reproducibility which would have complicated clinical translation of these agents. Results validated this transformation not only as a successful strategy despite warnings of decreased solubility (not true), but provided a far greater molar relaxivity. We reported a 5-fold enhancement over the prior technology while concurrently decreasing the actual physical amount of Gd(III) conjugated to the dendrimer by 65% further increasing the safety margin. The impact of this result reaches across to all macromolecular MR contrast agents regardless of platform to fully address safety, characterization, and reproducibility thereby furthering an entire fields potential for clinical translation of such agents. The exquisite advantages of dendrimer based agents over low molecular weight agents, e.g. Magnevist, continues to be very clearly demonstrated. In parallel to this improvement to abrogate toxicity concerns that has resulted in superior dendrimer based agents, the ability to finally move forward with actively targeted dendrimer based contrast agents that are discrete characterized agents was achieved through conjugation of a cystamine core dendrimer derived dendron conjugated in a 1:1 form with an antibody fragment. While successful, both isolation and purification were challenging with the dendrimer generation employed as well as relatively lower relaxivity results indicating the need to move onwards to a higher dendrimer generation. This investigation continues in parallel with the creation of a PEG appended trifunctional imaging agent that will permit incorporation of a radiological probe (PET imaging) with an optical probe. Patent applications continue to be prosecuted in parallel to publications revealing this technology to the field which should prove to make it yet more valuable to HHS and should also 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 terminated due to a lack of cooperation and access to instrumentation residing therein in what was to be a resource for all NCI researchers despite agreements to the contrary. However, established collaborations with Radiology, CC, the PET Dept, CC, NIMH, and extramural researchers at Johns Hopkins continue to be fruitful.