[99mTc]MAG3-mannosyl-dextran: a receptor-binding radiopharmaceutical for sentinel node detection
Introduction
Lectins [26] are receptors that recognized and bind specific carbohydrate bearing macromolecules. At the time of the first Receptor-Binding Radiotracer Workshop [10] only a small number of mammalian lectins have been identified. Figure 1 contains a list of mammalian lectins that we considered [35] as candidate targets for a receptor-binding radiotracer. These included lectins specific to hepatocytes [36], recticuloendothelial cells [25], myocardial cells [21], [37], and thrombin-activated platelets [14]. We selected the hepatic binding protein receptor as the molecular target for [99mTc]galactosyl-neoglycoalbumin [30]. A common theme of this early list was the simplicity of the carbohydrate specificity—all of the lectins recognized a monosaccharide. During the intervening twenty years lectins have been discovered with carbohydrate specificities of greater complexity. Some examples are: fucose-terminated tri-saccarharide-specific lectins involved in cell-cell interactions of the early mammalian embryo [34], and sialyl-terminated tetra-saccarharde-specific selectins, which recruit neutrophils to sites of endothelial inflammation [20]. Additionally, knowledge of their tissue distribution has been extended to include the immune system [33], the central and peripheral nervous system [45], and malignantly transformed cells [18].
Sentinel node biopsy is gradually replacing lymph node dissection in breast cancer patients [28]. The sentinel node is the first lymph node that receives drainage from a given anatomic area of the body [32]. Its location is determined by injection of a radiopharmaceutical [2] around the tumor site as well as a peritumoral injection of isosulfan blue dye [1], [15]. Lymphoscintigraphy is often performed preoperatively to localize the sentinel node on the day of surgery. The blue dye is typically injected at the beginning of surgery to facilitate the actual visualization of the sentinel node in the operative field. A multitude of studies have been performed whereby the sentinel node was removed and examined pathologically [29]. Patient’s in these studies then proceeded to standard levels I and II axillary node dissection. The sentinel node technique has been found to be an accurate predictor of lymph node status [4], [8], [17], with a false negative rate between zero and 12%. The sentinel node technique has also allowed for a more detailed pathologic evaluation of one specific lymph node via serial sectioning and immunohistochemistry. As such, micrometastatic disease is being discovered that previously was unrecognized. Therefore, the technique offers a smaller surgical dissection, minimal reduction in diagnostic accuracy compared to a standard axillary dissection, and the ability to focus detailed histopathological analyses to a single node [7], [43].
Numerous radiopharmaceuticals are presently utilized for sentinel node detection: Tc-99m-sulfur colloid, filtered Tc-99m-sulfur colloid, Tc-99m-albumin colloid, Tc-99m-antimony trisulfide, and Tc-99m-human serum albumin. The colloids, especially unfiltered Tc-99m-sulfur colloid, exhibit slow injection site clearance [16]. Consequently, scatter from the injection site can mask the sentinel node, which can often be only a few centimeters away. Tc-99m-human serum albumin, with no affinity for lymph node tissue, can travel through the lymph node chain, making sentinel node identification difficult. Because of the different agents in use, injection techniques are not standardized. The overall result is a long learning curve for both the radiologist and the surgeon, which can limit the success rate of sentinel node detection, and overall diagnostic accuracy.
This paper describes the continued development of a receptor-binding radiopharmaceutical for sentinel node detection. In 1998 we demonstrated the ability to image lymph nodes via the mannose binding protein receptor [40]. Recently, we described the synthesis of a dextran-based glycoconjugate, which displayed rapid injection site clearance [39]. Here, we present the synthesis and preliminary testing of a Tc-99m-labeled MAG3-mannosyl-dextran conjugate. Our goal is an instant labeling kit, which utilizes the high stability exhibited by the MAG3 chelation system.
Section snippets
Reagents
All aqueous solutions were prepared using deionized water (NANOpure Infinity, Barnstead-Thermolyne, Dubuque IA). Amino-terminated dextran was synthesized as previously described [39]. All dextran conjugates were lyophilized and stored at −80°C. Benzoyl-mercaptoacetylglycylglycyl-glycine (BzMAG3) was synthesized as described by Fritzberg et al. [13]. Cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-mannoside was synthesized by published methods [6], [27], [31]. Methanol was purchased as ACS grade,
Synthesis
The synthesis yielded a dextran conjugate with a mean mannose density of 21 mannose units per dextran molecule and a mean BzMAG3 density of 3 chelators per dextran. The mean molecular diameter was 5.5 ± 2.4 nanometers. The calculated molecular weight was 19,389 g/mole. The BzMAG3 density of the BzMAG3-dextran was not calculated.
Radiolabeling
Labeling of BzMAG3-dextran was quantitative; the electrophoresis scans displayed a single peak at the origin. An attempt to label amino-terminated dextran without BzMAG3
Discussion
To our knowledge this is the first demonstration that a receptor-binding radiotracer or a macromolecule can be directly labeled with Tc-99m via the MAG3 ligand system. Since the introduction of preformed chelates for technetium-99m labeling of antibodies [12] and antibody fragments [23], the high temperature and pH requirements of the DADS and MAG3 ligand systems have prevented the development of instant labeling kits. Consequently, the labeling of macromolecules, such as Fab [24] and Fv
Acknowledgments
This work was supported in part by National Cancer Institute Grant RO1-CA72751 and University of California Breast Cancer Research Program Grants 2RB-0018 and 4IB-0051. We thank Dr. Esther Lim for her participation in the rabbit biodistribution study, and Sudhakar Kasina, Ph.D. for his many helpful discussions and NMRs. We also thank the Neoprobe Corporation for the use of a Model 1000 portable radioisotope detector.
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