制备和评价<sup>99米</sup>锝标记的三齿螯合物预靶向使用生物正交化学
Summary
Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.
Abstract
Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.
Introduction
99m锝保留在核医学诊断中使用的主导放射性同位素，每年全球1，2，3进行超过5000万的成像程序。大多数临床使用99m锝剂是灌流型的放射性药物。有在其中99m锝涉及通过结扎特定生物标志物结合至靶向构建积极靶向化合物的数量有限。靶向99m锝放射性药物的创建通常是由99m锝-配体复合的对靶向分子的能力的影响阻碍了对感兴趣的生物标记物结合，或同位素半衰期不够长为具有更高分子量的生物分子用如抗体。后者通常需要数天的图像，以获得前的生物分子从非目标清除做卷烟的UE。预靶向提供了一种替代的方法来克服这些挑战。
预靶向结合生物正交化学已被证明是开发用于两个荧光和放射成像4，5，6，7，8个新的分子成像探针的有效方法。 -1,2,4,5-四（TZ）和反式 -cyclooctene（TCO）的衍生物之间的逆电子需求狄尔斯-阿尔德（IEDDA）反应， 如图1，已被证明是特别有效的6。这些组件的IEDDA反应可在PBS呈现快速动力学（K 2≈6000 M -1秒-1）和高选择性，因此非常适合在体内预先指定的应用9,10。
e_content“>使用的最常见的方法包括施用的TCO源性靶向载体和以下足够长的延迟时间，放射性标记的四嗪是基于11℃，（18）F，64的Cu，89 Zr和111在已经施用。放射性标记的四嗪报告11，12，13，14，15，相反，仅存在一个一个99m锝标记Tz的，其使用一个HYNIC型配位体需要使用共配体的，以防止在体内蛋白结合和降解制备的报告16。作为替代方案，我们在这里报告99m锝（Ⅰ）的合成中使用的是家族，其形成具有稳定的三齿络合物的配体的标记的四嗪[99m锝（CO）3] +核心。 <p class="jove_content" fo:keep-together.within-p年龄="“1”">
图1：四嗪和反 -cyclooctene之间的生物正交反应IEDDA。 请点击此处查看该图的放大版本。
制备配体的家族包含三齿螯合物，在极性和连接基团的金属结合区和Tz的（ 图2）之间的性质不同而不同。该目标是确定一个99m锝-四嗪构造，可以有效地定位和在体内的TCO标记位点和当未结合迅速明确反应，以产生高靶-非靶比率。为了测试配位体，二膦酸盐的TCO-衍生物（TCO-BP），使用17。之前我们已经表明，TCO-BP定位于活跃的骨代谢领域，可以与反应放射性四嗪在体内 18。它是一种方便的试剂测试新四嗪，因为它可以在一个单一的步骤进行制备和实验可以在本地化主要发生在关节（膝和肩）正常小鼠进行。
Protocol
Representative Results
Discussion
不同极性的四嗪联三齿螯合物的集合被制备，并且与在体内的TCO衍生物IEDDA反应其99m锝复合物的效用进行评价。一种有效的和可再现的99m锝标记方法是五四嗪螯合物，其中该配位体的浓度为10 -3 M的标记步骤之后是叔丁基基（化合物2-5）的脱保护的发展。配体的高浓度使用以提高放射化学产率，并减少反应时间，这减少了四嗪21的…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.
Materials
| Argon gas | Alphagaz | — | — |
| Na2CO3 | EMD Millipore | 106395 | — |
| Na2B4O7.10H2O | Anachemia | S9640 | — |
| KNaC4H4O6.4H2O | Anachemia | 217255 | — |
| Technelite 99mTc generator | Lantheus medical imaging | — | Source of 99mTcO4- |
| 0.9% Saline | Lantheus medical imaging | — | To elute generator |
| 1 M HCl | Lab Chem | — | — |
| MeOH | Caledon | — | — |
| ACN | Caledon | — | HPLC grade |
| Millipore H2O | Thermo Fisher Scientific | Barnstead Nanopure | — |
| DCM | Caledon | — | — |
| TFA | Caledon | — | — |
| PBS | Thermo Fisher Scientific | 10010023 | pH 7.4 1X |
| BSA | Sigma Aldrich | A7906 | — |
| Tween80 | Sigma Aldrich | P8047 | — |
| Isoflurane | CDMV | 108737 | Supplier: Fresenius Kabi Animal Health |
| HPLC | Waters | 1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590) | — |
| HPLC column for analysis and purification of compounds 2-4 | Phenomenex | 00G-4435-E0 | Gemini® 5 µm C18 110 Å, LC Column 250 x 4.6 mm, |
| HPLC column for analysis and purification of compounds 1 and 5 | Waters | 186003115 | XBridge BEH C18 Column, 130 Å, 5 µm, 4.6 mm X 100 mm |
| Microwave Reactor | Biotage | Initiator 8 | — |
| Biotage V10 Evaporator | Biotage | Serial # V1041 | — |
| Dose calibrator | Capintec, Inc. | CRC-25R | — |
| Gamma counter | Perkin Elmer | Wizard 1470 Automatic Gamma Counter | — |
| Animal room scale | Mettler Toledo | XP105 Delta Range | — |
| Microwave vials | Biotage | 355629 | 0.5-2 mL |
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