In vitro characterization of a novel prostate cancer therapeutic radiopharmaceutical

Objectives Androgen receptor (AR) is crucial for the development and progression of prostate cancer, including androgen independent and hormone refractory malignancies. 5α-Dihydrotestosterone (DHT) is an endogenous hormone that binds to AR with high affinity and specificity. Following its association with a ligand such as DHT, AR translocates to the nucleus and binds directly to DNA, making it especially suitable for radionuclides emitting short range, high linear energy transfer (LET) radiations such as in the Auger process following electron capture or internal conversion radioactive decay. This work characterizes the in vitro AR binding affinity and chemical stability of 7α-[^80mBr]bromo-5α-dihydrotestosterone (^80mBrDHT), a DHT analogue labeled with Auger-emitting ^80mBr (t_1/2 = 4.4 h), for use as a new targeted radionuclide therapeutic agent for prostate cancer.

Methods Radioactive and non-radioactive BrDHT were prepared from a commercially available chemical precursor. The relative AR affinity of non-radioactive BrDHT compared with unmodified DHT was assessed using a time-resolved fluorescence resonance energy transfer assay (TR-FRET, Thermo Fisher Scientific Life Sciences Solutions, Madison, WI) using R1881 as a known AR agonist control. Radiobromine ([asterisk]Br, [asterisk] = 76,77,80m,82) used to synthesize [asterisk]BrDHT was produced through the proton irradiation of nickel selenide and chemically isolated through dry distillation. [asterisk]BrDHT was tested for chemical stability in water at 20°C, phosphate-buffered saline (PBS) with 5% bovine serum albumin (BSA) at 37°C, and 100% whole mouse serum at 37°C using silica thin-layer chromatography (TLC) with 1 : 1 :: dichloromethane : ethyl acetate as mobile phase. The radiobromine distribution on developed TLC plates was visualized using storage phosphor autoradiography.

Results Plotting the percent activation of AR as a function of ligand concentration allows for the assessment of relative AR affinity of BrDHT and DHT, as shown in figure 1, bottom left. These data are summarized by two sigmoidal curves with the half maximum effective concentration (EC₅₀) for BrDHT measured to be 11 nM and DHT to be 4.0 nM, while measuring an EC₅₀ for R1881 of 1.4 nM. The chemical debromination of [asterisk]BrDHT, shown in Figure 1 bottom right, was observed to occur in water, 5% BSA solution, and mouse serum with a degradation half-life of ~8 hours, approximately twice that of the nuclear decay half-life for the radiotherapeutic isotope, ^80mBr.

Conclusions The in vitro characterization of [asterisk]BrDHT demonstrates its nanomolar-level AR affinity and adequate chemical stability. These promising results warrant further investigation into the Auger-induced cellular toxicity of ^80mBrDHT in AR-positive and AR-negative cell lines and determination of the biodistribution and in vivo stability of [asterisk]BrDHT through preclinical PET imaging of ⁷⁶Br. Through this work, the potential of localized, high LET Auger electrons may be realized in this novel prostate cancer targeted radionuclide therapeutic agent. $$graphic_031EA368-646F-47B1-A02A-AE9B4695D80A$$ Research Support:This work was supported in part by a University of Wisconsin Institute for Clinical and Translational Research Pilot Award