Pre-clinical Evaluation of Iodoazomycin Arabinofuranoside (IAZA) and Fluoroazomycin Arabinofuranoside (FAZA) as Hypoxia-targeting Therapeutic Agents in a Head and Neck Cancer Model

• Author / Creator

  Rashed, Faisal B

• Tumour hypoxia is a well-recognized clinical problem that promotes resistance to anti-cancer treatment (chemotherapy, radiotherapy, and immunotherapy), increases rates of recurrent disease, and ultimately leads to poor patient outcome. Eradication of hypoxic tumours is, therefore, essential for effective management of solid malignancies. Targeting hypoxic tumours requires a precision approach using compounds, such as nitroimidazoles (NIs), that are structurally equipped to reach and become activated within diffusion-limited tumour niches. NIs undergo bioreductive metabolism only under hypoxia and subsequently bind to cellular constituents, trapping the drug inside hypoxic cells. The selective accumulation of NIs has been successfully exploited for hypoxia diagnosis (either by radiological imaging or through immunological detection). However, their efficacy as anti-cancer agents is limited. Except for nimorazole, most NIs failed to provide definitive clinical advantage. Unfortunately, considerable gaps still remain in our understanding of their mechanism of action at the cellular level, which makes it challenging to further improve upon these compounds. The present study aimed to characterize the molecular phenotype induced by two 2-NI compounds, iodoazomycin arabinofuranoside (IAZA) and fluoroazomycin arabinofuranoside (FAZA), which are clinically validated hypoxia-selective radiotracers with promising therapeutic potentials. A major focus of this study was to identify and analyze the molecular targets of 2-NIs in order to elucidate their potential mechanism(s) of action.
  Chapter 2 describes the synthesis and applications of azidoazomycin arabinofuranoside (N3-AZA), a novel click-chemistry compatible analogue of IAZA and FAZA, designed to facilitate (a) proteomic analysis of 2-NI targeted proteins in FaDu head and neck cancer cells, and (b) rapid and efficient labelling of hypoxic cells and tissues. A total of 62 target proteins were identified; bioinformatic analysis revealed that many of them participate in key canonical pathways including glycolysis and hypoxia-inducible factor 1α (HIF1A) signaling that play important roles in the cellular response to hypoxia. Proteins such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase P (GSTP1) appeared as top hits, and N3-AZA adduct formation significantly reduced their enzymatic activity selectively under hypoxia. Fluorescent staining of N3-AZA-adducts also supported cell and tissue imaging for mapping tumour hypoxia, with comparable results to the current gold standard reagent, pimonidazole. However, because our approach uses click chemistry rather than immune-detection, the process is considerably less time-consuming and more cost-effective.
  Chapter 3 provides a detailed analysis of the cellular phenotype induced by IAZA and FAZA under hypoxia. Hypoxic cells displayed higher sensitivity to IAZA and FAZA treatment, where the drugs induced cytostasis by compromising DNA replication, slowing down cell cycle progression and inducing replication stress. Effects of IAZA and FAZA on target cellular macromolecules (DNA, proteins and glutathione) were characterized. Covalent binding of NIs was only observed to cellular proteins, but not to DNA. While protein levels remained unaffected, catalytic activities of 2-NI target proteins, such as GAPDH and GSTP1 were significantly curtailed in response to IAZA/FAZA treatment under hypoxia. Single intraperitoneal (i.p.) injection of IAZA was well tolerated in mice (up to 600 mg/kg body weight), slowed down initial tumour growth, and reduced hypoxia levels in subcutaneous FaDu tumours.
  Hypoxia selective radiosensitization capacities of IAZA and FAZA were explored in Chapter 4. Low dose radiation treatment with these 2-NI compounds mostly generated additive effects under hypoxia. However, synergy was observed when hypoxic cells were treated with radiation doses >12 Gy (for IAZA) or >18 Gy (for FAZA). Combination treatment enhanced radiation induced DNA damage under hypoxia, observed through γ-H2AX immunocytochemistry and alkaline comet assay. No additional therapeutic benefit was observed when mice bearing subcutaneous FaDu tumours were injected i.p. with IAZA (400 mg/kg body weight) and irradiated with a single 10 Gy radiation dose. Given that chemotherapeutic agents are usually administered as multiple doses, it is worth exploring the effects of multi-dose treatment of IAZA on radiation response.
  An overall discussion of the results and suggestions for future research is included in Chapter 5. Also, since 131I-IAZA has potential for systemic radiotherapy, a preliminary toxicity analysis of non-radioactive IAZA administered in mice via an intravenous (i.v.) mode is provided, which indicates i.v. administration of IAZA is non-toxic. Established clinical efficacy of IAZA as a hypoxia imaging agent together with these data will support future in vivo experiments exploring IAZA as a potential theranostic agent.

• Subjects / Keywords

  • Hypoxia
  • Nitroimidazole

• Graduation date

  Spring 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-nvgd-rw85

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.