Development of ¹³¹I-ixolaris as a theranostic agent: metastatic melanoma preclinical studies

Tissue factor (TF), a blood coagulation protein, plays an important role in tumor growth, invasion, and metastasis. Ixolaris, a tick-derived non-immunogenic molecule that binds to TF, has demonstrated in vivo inhibitory effect on murine models of melanoma, including primary growth and metastasis. This work aimed to: I) develop an efficient and stable labeling technique of ixolaris with Iodine-131(¹³¹I); II) compare the biodistribution of ¹³¹I and ¹³¹I-ixolaris in tumor-free and melanoma-bearing mice; III) evaluate whether ¹³¹I-ixolaris could serve as an antimetastatic agent. Ixolaris radioiodination was performed using iodogen, followed by liquid paper chromatography. Labeling stability and anticoagulant activity were measured. Imaging studies were performed after intravenous administration of free ¹³¹I or ¹³¹I-ixolaris in a murine melanoma model employing the B16-F10 cell line. Animals were divided in three experimental groups: the first experimental group, D0, received a single-dose of 9.25 MBq of ¹³¹I-ixolaris at the same day the animals were inoculated with melanoma cells. In the second group, D15, a single-dose of 9.25 MBq of ¹³¹I-ixolaris or free ¹³¹I was applied into mice on the fifteenth day after the tumor induction. The third group, D1-D15, received two therapeutic doses of 9.25 MBq of ¹³¹I-ixolaris or ¹³¹I. In vitro studies demonstrated that ¹³¹I-ixolaris is stable for up to 24 h and retains its inhibitory activity on blood coagulation. Biodistribution analysis and metastasis assays showed that all treatment regimens with ¹³¹I-ixolaris were effective, being the double-treatment (D1/D15) the most effective one. Remarkably, treatment with free ¹³¹I showed no anti-metastatic effect. ¹³¹I-ixolaris is a promising theranostic agent for metastatic melanoma.