A synthetic macromolecule for sentinel node detection: Tc-99m-DTPA-mannosyl-dextran

We report the synthesis and preliminary biologic testing of a synthetic mac romolecule, Tc-99m-diethylenetriaminepentaacetic acid (DTPA)-mannosyl-dextr an, for sentinel node detection. Methods: Synthesis started with a P-step p rocess that attaches a high density of amino-terminated leashes to a dextra n backbone. Allyl-bromide was reacted with pharmaceutical-grade dextran to yield allyl-dextran. After diafiltration with water, filtration, and lyophi lization, the product was reacted with aminoethanethiol and ammonium persul fate. The resulting amino-conjugated dextran was dialyzed, filtered, and ly ophilized. The mixed anhydride method was used to attach DTPA; after dialys is, filtration, and lyophilization, 2-imino-2-methoxyethyl-1-D-mannose was used to attach the receptor substrate. The molecular diameter was measured by dynamic light scattering. Amino, mannose, and DTPA densities were measur ed by trinitrobenzene sulfonate assay, sulfuric acid/phenol assay, and indu ctively coupled plasma spectroscopy of gadolinium-DTPA-mannosyl-dextran, re spectively. Receptor affinity was measured by Scatchard assay of rabbit liv er. Axillary, popliteal, and iliac lymph nodes and each injection site were assayed for radioactivity at 1 and 3 h after injection of approximately 3. 7 MBq (0.050 mt) Tc-99m-DTPA-mannosyl-dextran (0.22 nmol) or filtered Tc-99 m-sulfur colloid into the foot pads. Four animals were studied at each time point. Results: DTPA-mannosyl-dextran had a molecular weight of 35,800 g/m ol and a molecular diameter of 7.1 nm. The final amine, mannose, and DTPA d ensities were 23, 55, and 8 mol per dextran. Labeling yields were in excess of 98% and stable for 6 h. Specific activities of 74 x 10(6) GBq/mol were achieved. The equilibrium dissociation constant for binding to the mannose- terminated glycoprotein receptor was 0.12 +/- 0.07 nmol/L. The popliteal ex traction at both 1 h and 3 h was significantly (P < 0.05) higher for Tc-99m -DTPA-mannosyl-dextran (90.1% +/- 10.7% and 97.7% +/- 2.0%, respectively) t han for filtered Tc-99m-sulfur colloid (78.8 +/- 6.5 and 67.4% +/- 26.8%, r espective ly). Tc-99m-DTPA-mannosyl-dextran exhibited significantly faster injection site clearance than did filtered Tc-99m-sulfur colloid. The Tc-99 m-DTPA-mannosyl-dextran percentage injected dose (%ID) for the front and re ar paws was 52.6 +/- 10.5 and 52.3 +/- 8.0 at 1 h and 45.7 +/- 8.5 and 43.6 +/- 8.2 at 3 h after administration. The filtered Tc-99m-sulfur colloid %I D for the front and rear paws was 70.4 +/- 11.0 and 66.3 +/- 15.1 at 1 h an d 55.5 +/- 7.8 and 66.9 +/- 8.5 at 3 h. Lymph node accumulation of each age nt at either 1 or 3 h was not significantly different. Conclusion: Tc-99m-D TPA-mannosyl-dextran is a receptor-based sentinel node radiotracer that exh ibits the desired properties of rapid injection site clearance and low dist al node accumulation. This molecule is the first member of a new class of d iagnostic agents based on a macromolecular backbone with a high density of sites for the attachment of substrates and imaging reporters.