Molecular imaging and targeted therapy of estrogen receptor expressing tumors using radiolabelled non-steroidal synthetic antiestrogenic drug

Objectives: Estrogen receptor (ER) expressing tumor show better prognosis due to responsiveness to anti-estrogen treatment. Tamoxifen is an FDA approved anti-estrogenic drug. Radiolabelling of tamoxifen with Tc-99m and Re-188 as a potential theranostic pair for ER expressing breast cancer patients.

Methods: Tc-99m and Re-188 tricarbonyls were synthesized in house using sodium boranocarbonate as in situ carbon monoxide producing source. For Re-188 tricarbonyls borane ammonia and phosphoric acid were also added. Tamoxifen was radiolabelled via Tc-99m and Re-188 tricarbonyl core. QC was performed and complexes were also characterised by MALDI-TOF and NMR. In-vitro receptor binding and blocking studies were performed in cell lines (MCF-7, MDA-MB-231) with Tc-99m tricarbonyl tamoxifen. MTT assay was done to assess cytotoxicity of Re-188 tricarbonyl tamoxifen. Biodistribution studies were performed in female wistar albino rats with Tc-99m tricarbonyl tamoxifen. Patient imaging was done after obtaining ethical clearance from the institute. Four histopathological proven ER expressing patients were recruited and F-18 FDG PET/CT was performed. The whole body and SPECT/CT were acquired after injection of Tc-99m tricarbonyl tamoxifen (8-14 mCi) and the scans were compared with F-18 FDG PET/CT for tracer uptake in the lesion/s

Results: Tc-99m and Re-188 tricarbonyl and Tc-99m and Re-188 tricarbonyl tamoxifen were synthesized with high radiolabelling efficiency (>99%). The formulations were found sterile and apyrogenic. In mass spectra, a major peak corresponding to the sum of molecular weights of Tc-99m/Re-188 tricarbonyl and tamoxifen (M+H)⁺ indicated good radiolabelling. The chemical shift observed in the protons of α'-CH₂ and β'-CH₂ and N(CH₃)₂ in NMR spectra of Re-188 and Tc-99m tricarbonyl tamoxifen suggested binding of Tc-99m/Re-188 tricarbonyl near aliphatic side chain of tamoxifen. Maximum 30% binding was observed in estrogen expressing MCF-7 cell line with respect to ~3% binding with non estrogen expressing MDA-MB-231. Receptor binding (%) was reduced to 8.4% when preincubated with excess tamoxifen. Re-188 tricarbonyl tamoxifen (40µCi, 2µg) showed 93.2% cytotoxicity on MCF-7 cells (5X10³). More than 90% cytotoxicity was observed with 1/20 times amount of tamoxifen in Re-188 tricarbonyl tamoxifen as compared to tamoxifen alone (20µg, 71.48%) and Re-188 alone (40µCi, 36.9%). In biodistribution study in rats, counts were noted in lung, liver, spleen and kidney. High blood counts were noted up to 2 hrs. In clinical study, Tc-99m tricarbonyl tamoxifen uptake was observed at primary site (breast) and lymph nodes in three patients. On comparison, Tc-99m tricarbonyl tamoxifen SPECT/CT findings were in concordance with F-18 FDG PET/CT. However, the low lesion to background contrast was observed with Tc-99m tricarbonyl tamoxifen. No uptake of Tc-99m tricarbonyl tamoxifen was seen in fourth patient, who was on tamoxifen therapy from last six months, probably due to receptor saturation However, lesions were visualised on F-18 FDG PET/CT image.

Conclusions: Tc-99m tricarbonyl tamoxifen could be a potential diagnostic tool for ER expressing breast cancer patients in centres not having on site cyclotron facility. The radiopharmaceutical shows the therapeutic potential when Re-188 is used in lieu of Tc-99m.

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