BIUX2X2

Objectives: With the objective to develop a potential radiopharmaceutical for estrogen receptors imaging we have prepared and evaluated an estradiol derivative sodium(1-carboxy-2-(4-((13S,17S)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl)-1H-1,2,3-triazol-1-yl)ethyl)carbamodithioate (L), labelled with ^99mTc. Labelling was performed through the preparation of a symmetric ^99mTc(V)-nitrido complex due to the intrinsic structural robustness of this type of compounds.

Methods: Ethynilestradiol (144 mg-0.48 mmol) was derivatized to introduce an amino group by means of a Click Chemistry reaction with N-Boc-azidoalanine (200 mg-0.48 mmol) .The product was deprotected and the dithiocarbamate introduced by reaction with carbon disulphide (0.2mL-3.32 mmol) and sodium hydride (35 mg-1.46 mmol) at 65°C for 1,5 hours. The product (L) was characterized by spectroscopic techniques. Labelling was performed in two steps: preparation of the ^99mTc-nitrido precursor by reduction of pertechnetate (20-50mCi, 1mL) in the presence of succinic dihydrazide (5mg, 0.5mL) followed by substitution achieved by incubating L (3mg, 300 µL methanol) with 0.1 mL of the nitrido precursor for 30 minutes a 65°C (5-14 mCi). Radiochemical purity (RP) was assessed by reverse phase HPLC. Stability of the labelled compound in reaction milieu and in human plasma (at 37 °C) was also studied. Binding to plasma proteins was determined by size exclusion chromatography and lipophilicity was measured by means of the octanol/phosphate buffer pH=7.4 partition coefficient. Biodistribution in normal Sprague Dawley rats (female, 3 animals per group) was studied at 30, 60 y 120 min post-injection. In vitro studies were performed on MCF7 cells. The% binding of estrogen receptors was determined, as this binding varies according to activity and time and% efflux.

Results: The ligand L bears a dithiocarbamate group that provides two electron-donor atoms to bind to Tc in the form of a Tc (V) nitride complex, resulting in a symmetrical complex with two molecules of L. Labelling was successfully performed. The precursor and the Tc complex were obtained with RP of 100%. The complex was stable in reaction milieu and in human plasm for at least 3 hours (RP=95%). It showed a protein binding of 40% and a log P of 0.7±0.1. Biological evaluation in normal animals showed high liver activity (14.8 ± 2.5%) and excretion trough the gastrointestinal tract (29.97±5.58%). Activity in other organs or tissues was very low. The % of blood activity decreases as biodistribution time increases. The maximum % binding to estrogen receptors was 6.1% for 20 μCi. This % of binding did not vary with time (4 hours). The % efflux (representing the amount of complex released from receptor) was 70% after one hour and did not increase with time.

Conclusion: A novel estradiol derivative was obtained and radiolabelled by preparing a symmetric Tc(V)-nitride complex. The product has a high RP and good stability. Physicochemical properties are adequate. The cell uptake study revealed that the complex obtained binds strongly with ER receptors and the cell efflux study showed only 30% of the complex remains tightly bound to the estrogens receptors. In vivo studies are needed with animals bearing breast tumors that overexpress estrogen receptors. Acknowledgements ANII, Pedeciba-Química, CHLCC, Bayer-Schering Pharma AG-Uruguay