Hypoxia-activated prodrugs: paths forward in the era of personalised medicine

Abstract

Tumour hypoxia has been pursued as a cancer drug target for over 30 years, most notably using bioreductive (hypoxia-activated) prodrugs that target antineoplastic agents to low-oxygen tumour compartments. Despite compelling evidence linking hypoxia with treatment resistance and adverse prognosis, a number of such prodrugs have recently failed to demonstrate efficacy in pivotal clinical trials; an outcome that demands reflection on the discovery and development of these compounds. In this review, we discuss a clear disconnect between the pathobiology of tumour hypoxia, the pharmacology of hypoxia-activated prodrugs and the manner in which they have been taken into clinical development. Hypoxia-activated prodrugs have been evaluated in the manner of broad-spectrum cytotoxic agents, yet a growing body of evidence suggests that their activity is likely to be dependent on the coincidence of tumour hypoxia, expression of specific prodrug-activating reductases and intrinsic sensitivity of malignant clones to the cytotoxic effector. Hypoxia itself is highly variable between and within individual tumours and is not treatment-limiting in all cancer subtypes. Defining predictive biomarkers for hypoxia-activated prodrugs and overcoming the technical challenges of assaying them in clinical settings will be essential to deploying these agents in the era of personalised cancer medicine.

Figure 1

Molecular pharmacology of HAPs. Aromatic nitro (e.g. evofosfamide, tarloxotinib, PR-104, nitroCBI) and N-oxide (e.g. tirapazamine, SN30000) HAPs are activated in tumours by a process that is initiated by enzymatic one-electron reduction to yield a prodrug radical anion. In well-oxygenated tissue, the unpaired electron is rapidly scavenged from this reducing radical by molecular oxygen, resulting in a futile redox cycle. In the absence of oxygen, the radical anion either fragments or is reduced further to produce a cytotoxic effector species that engages a pharmacological target (e.g. by kinase inhibition or damaging DNA through alkylation, oxidation or poisoning of topoisomerase II). Collectively, this understanding of the molecular pharmacology of HAPs implies that presence of tumour hypoxia, expression of one-electron reductases in addition to cell-intrinsic modifiers of sensitivity of the cytotoxic effector may influence the efficacy of HAPs in individual malignancies.

Figure 2

HAP activation in tumours. (A) Identification of POR as a major determinant of SN30000 sensitivity by high-throughput shRNA screening of HT-29 and PANC-1 cells. The metric plotted is the Z-transformed log₂ of hairpin enrichment factor (treated/control) following exposure to SN30000. (B) Immunohistochemical staining for POR in a tissue section from an HPV-negative hypopharyngeal squamous carcinoma. Intense, relatively homogenous POR staining is observed in the malignant epithelium with minimal reactivity in the tumour-associated stroma. (C) Pre-treatment [¹⁸F]-misonidazole PET imaging for hypoxia in the same patient showing an avid mass in the posterior pharynx. Reproduced, with permission, from (Hunter et al, 2015).

Figure 3

Hypothetical transverse section of tumour vascular chords showing steady state hypoxia gradients resulting from spatial heterogeneity of perfusion. Therapeutically significant zones are indicated in the oxygen concentration gradient, demarking cells that are classified as euoxic (O₂>25 μM), moderately hypoxic (O₂<25 μM, typically sufficient to induce a HIF-1 response), radiobiologically hypoxic (O₂<5 μM) and essentially anoxic (O₂<0.5 μM, enabling efficient activation of nitroaromatic HAPs) beyond which necrosis ensues. Numbers indicate intravascular O₂ concentrations (μM), which can vary temporally as well as spatially. Perivascular hypoxia is shown associated with two poorly perfused vessels in the centre of the section. Chemoresistant and radioresistant cells at moderate hypoxia (‘HIF activation'+‘radiobiological hypoxia') are accessible to bystander effects resulting from diffusion of effectors from nitroaromatic HAPs (arrows), but only if contiguous with severely hypoxic (anoxic) regions. Hypoxic regions that are beyond the bystander range of nitroaromatic prodrugs (asterisks) may be more effectively treated by HAPs such as benzotriazine di-oxides that are less sensitively inhibited by oxygen.