L. I. Banla1, J. Quintana2, S. Olberg3, T. S. C. Ng4, J. P. Schuemann3, K. D. Held5, W. L. Hwang6, and M. A. Miller2; 1Harvard Radiation Oncology Program, Boston, MA, 2Center for Systems Biology, Massachusetts General Hospital, Boston, MA, 3Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA, 4Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 5Massachusetts General Hospital, Harvard Medical School, Boston, MA, 6Harvard Radiation Oncology Program, Massachusetts General Hospital, Boston, MA
Purpose/Objective(s):Radicals generated by ionizing radiation (IR) can drive specific chemical reactions. This phenomenon underpins radiation-dependent prodrugs (RPDs), which are specifically activated by IR. RPDs thus represent a novel approach to enhance the therapeutic index of antineoplastic drugs by harnessing the spatial targeting potential of radiotherapy. Independent research groups have shown the effectiveness of RPDs in various animal tumor models, highlighting their translational potential. Despite the expanding array of IR-sensitive linkers, critical questions remain. In this work, we investigate the impact of clinically relevant factors including radiation energy and dose rate on cleavage efficiency across distinct linker chemistries. Materials/
Methods: We confirmed the chemical structures of radio-conversion substrates via NMR and mass spectrometry. Radiation-induced activation was examined across four linker chemistries: dimethoxy benzyl alcohol (DMBA), phenyl azide (Az), amine oxide (NO), and quaternary ammonium (QA), all integrated with a common quenched fluorochrome core structure for standardized comparison and sensitive detection. Gentisic acid served as the reducing radical quencher, with tert-butanol and sulfuric acid as the oxidizing counterpart. We evaluated radiation-induced conversion efficiency by quantifying the dose-dependent release of probes across various radiation doses and conditions. Radiation was applied using a 320kV X-ray irradiator, and a 6MV clinical beam, with dose rates ranging from 10 to 600 cGy/min. Solid water phantoms (5 and 10 cm) simulated treatment depth. Payload release was measured by calibrated LC/MS analysis of product mixtures post-irradiation. Results: Using radical species quenchers, we determined the specific radical species dependence for each probes radioconversion. The DMBA conversion was driven by oxidizing radicals, whereas the Az, NO, and QA linkers primarily depended on reducing radicals, aligning with both existing literature and predicted reaction mechanisms. Our evaluation of conversion efficiencies revealed two key insights: first, linkers reliant on reducing radicals demonstrated superior conversion efficiencies (69.6 nM/Gy for DMBA vs 131-183 nM/Gy for Az, NO and QA); second, across a spectrum of radiation dose rates, energies, and treatment depths, conversion efficiencies remained comparable for each linker chemistry. Importantly, unirradiated linker stability was variable across linker moieties, highlighting the potential need to balance sensitivity and specificity in RPD activation. Conclusion: Our results highlight linker chemistry and radical-species dependence as particularly important factors in determining conversion efficiency. Moreover, we note comparable conversion efficiencies across a range of radiation conditions extending to those of clinical relevance. This work offers insight into the design of future RPDs and their translation into clinical studies.
.jpg "Leou Ismael Banla, MD, PhD photo")