Hedgehog derivatives for tumor imaging

Objectives Abnormal activation of the stem cell pathway Hedgehog (HH) has been observed in several cancers. Due to a positive feedback loop, the HH receptor Patched (PTCH) is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH ligand sonic (SHH) for detection of cancer cells by PET and SPECT.

Methods DOTA-SHH was prepared by coupling of DOTA-NHS to the 19.5 kDa human N-terminal SHH protein. The conjugate was analyzed by MALDI-MS, ESI-MS and reverse-phased high performance liquid chromatography (RP-HPLC) and labeled with 68Ga for PET and 111In and 177 Lu for SPECT. Radiochemical purity was assessed by radio-TLC and radio-HPLC. In vitro bioactivity was evaluated by receptor binding studies in breast (MCF-7, BT474 and MDA-MB-231), prostate (PC3 and DU145) and lung cancer (A549) cell lines. Studies were performed with or without blocking with cold SHH. PTCH receptor expression and receptor binding of SHH was evaluated in the presence of the HH pathway inhibitor cyclopamine.

Results DOTA-SHH was obtained with in yields of 45% - 60% and purity > 90%. Radiolabeling of the conjugate with 68Ga and 177Lu was accomplished with a radiochemical purity of > 98% and specific radioactivity of 4.3E+05 MBq/gram (68Ga) and 1.26E+02 MBq/gram (177Lu) and radiochemical yields > 95%. High receptor binding was observed in cancer cells and cellular uptake correlated with PTCH expression levels. Wash-out studies showed retention of over 80% of radioactivity after 4 hours. Addition of 100-fold excess cold SHH reduced cellular uptake by approximately 90%. Pretreatment with cyclopamine significantly reduced PTCH expression and SHH receptor binding.

Conclusions These findings demonstrate that radiometalated SHH can be synthesized in high yield and specific radioactivity with retention of receptor-mediated binding. Compounds of this class may provide a method to follow response to HH-targeted therapies