Synthesis and characterization of In-111-DTPA-N-TIMP-2: A radiopharmaceutical for imaging matrix metalloproteinase expression

The matrix metalloproteinases (MMPs) are enzymes involved in the turnover o f the extracellular matrix. Their overexpression in tumors is implicated, i n the metastatic process and may provide a target for diagnostic tumor imag ing by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tis sue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential tar geting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to p roduce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was co njugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA-N-TIMP-2 conjugate (identified by electrospra y ionization mass spectrometry) was isolated by anion-exchange chromatograp hy. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine- 116, which is distant from the site of MMP interaction. The conjugate was r adiolabeled with indium-111 to give In-111-DTPA-N-TIMP-2 with a specific ac tivity of at least 4 MBq/mug and a radiochemical yield and purity of > 95%, by incubation with (IUCl3)-I-111, without need for postlabeling purificati on. The product was sterile, pyrogen-free, and stable in serum over 48 h an d retained full inhibitory activity in a fluorimetric binding assay. With t hese attributes, In-111-DTPA-N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.