Development of Hypoxia-Selective Trapping Agents for Targeting Prostate Cancer

Objectives Gastrin-releasing peptide receptor (BB2r) is overexpressed in various cancers including prostate cancer. Bombesin (BN) peptides as targeting agents for the BB2r-positive cancers have been widely explored. However, one of the challenges of BN based theranostic agents is their fast clearance from tumors. Prostate cancers, like most solid tumors, contain significant regions of hypoxia due to disordered vasculature. We are currently exploring the utility of hypoxia selective trapping agents (HSTA) such as 2-nitroimidazole acetic acid (2-NIAA) and hypoxia activated nitrogen mustards, that irreversibly bind to macromolecules in hypoxic cells, to enhance the residence time of BB2r-targeted therapeutic agents in prostate cancer tumors. Herein, we describe our progress in the synthetic development and mechanistic investigation of HSTA enhanced BN conjugates.

Methods An alkylating phosphoramidate HSTA has been designed and synthesized. The key intermediate, N-(2-bromoethyl)prop-2-yn-1-amine (AM),was synthesized from ethanolamine, propargyl bromide as starting materials. The prodrug was achieved by stepwise introduction 2-bromoethylamine, AM and 2-nitroimidazoles to POCl3. To characterize the nitrogen mustard analog, we used 1H and 31P NMR spectroscopy or X-ray crystallography. BN-conjugates incorporated with 2-NIAA were synthesized and labeled with either 177/natLuCl3 or Cy5 fluorophore. To investigate adduct formation of these agents, 2-NIAA alone or natLu-conjugates were either mixed with glutathione in the presence of Zn-NH4Cl or with NADPH and cytochrome c, under hypoxic conditions. To explore hypoxia selectivity of BN-conjugates, adjacent tumor sections from PC-3 xenograft mice injected with177Lu Labeled BN-conjugates were stained with pimonidazole and were exposed to generate autoradiograph. To study cell trafficking, endosomes and lysosomes were fluorophore labeled in PC-3 cells.

Results A Phosphoramidate analogue containing a nitrogen mustard structure was synthesized. The 2-nitroimidazole incorporated BB2r-targeted conjugates upon reduction formed adducts with free thiols. Based on the analog utilized and the conditions employed, the percentage of adduct formation varied from 8% to 13%. Co-localization of the tumor hypoxia and autoradiography 24h after radio-labeled conjugate injection confirmed the selectivity of BN-conjugate hypoxia trapping ability. Cell trafficking studies showed cellular uptake and travel from early endosomes to lysosomes after 2h incubation.

Conclusions The adduct formation of the BB2r-targeted conjugates with free thiols implies the possible trapping mechanism in hypoxic cells. BN-conjugates incorporated with 2-nitroimidazole indeed have hypoxia selectivity.