Abstract SY34-01: New prodrugs for targeting tumor hypoxia
Authors: Wilson William Robert 
Published: May 2019

Abstract
Introduction
Hypoxia, frequently considered a hallmark of cancer, is a consequence of the inefficient vascularisation of tumors. In a number of tumor types hypoxia contributes to tumor progression and is associated with poor prognosis and resistance to therapeutic agents, providing a strong rationale for targeting hypoxia. However, preclinical and clinical investigation of a diverse range of hypoxia-activated bioreductive prodrugs (quinones, nitro compounds, N-oxides, transition metal complexes) over three decades has demonstrated the goal of exploiting hypoxia with small molecule drugs to be an elusive one. Despite this, there is now clinical proof-of-principle in the demonstration that PET imaging of hypoxia identifies a subset of patients who benefit from addition of the hypoxia-selective benzotriazine di-N-oxide prodrug tirapazamine (TPZ) to cisplatin chemoradiation for head and neck cancer (1), although this requires confirmation in larger multi-center studies. Just as importantly, experience with TPZ and other clinically-evaluated bioreductive prodrugs (porfiromycin, AQ4N, PR-104, TH-302) has identified challenges which need to be met in the development of more useful hypoxia-targeting agents.
Finessing hypoxia as a target: high or low K values for oxygen?
Many of these challenges can be traced to the problem that hypoxia is a quantitative rather than qualitative target; oxygen gradients are present in some normal tissues, and physiological levels of hypoxia appear to be sufficient to activate bioreductive drugs such as TPZ (2,3). Further, selectivity for tumor hypoxia can be compromised if metabolic consumption of prodrugs under hypoxia is fast enough to limit their penetration, as occurs with TPZ (4), although tools for designing prodrugs with improved extravascular diffusion are becoming available (5). An additional strategy we have explored for overcoming both these problems (activation under physiological hypoxia, and penetration into hypoxic tissue) is to confine metabolic activation to very low oxygen concentrations unique to tumors. This is achievable with dinitrobenzamide mustard prodrugs such as PR-104, which have K values for O2 ~ 10-fold lower than TPZ (6, 7). The water-soluble phosphate ester PR-104 is converted in vivo to the alcohol PR-104A, which is reduced in tumors to hydroxylamine PR-104H, an activated DNA crosslinking agent (8-10). Local diffusion of PR-104H provides “bystander” killing of cells at higher pO2 in tumors, enabling killing of more moderately hypoxic cells to be dependent on severe hypoxia in tumors. However, this comes at the price of a smaller pool of activating cells. Given that tumor xenografts tend to be more hypoxic than tumors in patients, it is not yet clear whether this trade-off (more selective activation in fewer cells) will be beneficial.
Identifying and profiling prodrug-activating reductases
An allied problem is that many bioreductive prodrugs are activated in parallel by oxygen-independent two-electron reductases (2eR) such as DT-diaphorase that bypass the oxygen-sensitive free radical intermediate generated by one-electron reductases (1eR). This “off-mechanism” 2eR activation pathway may be therapeutically useful if the 2eR responsible are selectively expressed in tumors. Until recently, bioreductive prodrug activation by endogenous 2eR has been considered largely restricted to the quinones, but we have shown PR-104A is a substrate for 2e nitroreduction by aldo-keto reductase 1C3 (AKR1C3), which is also overexpressed in some tumor types (11). However there is evidence for expression of AKR1C3 in human myeloid progenitor cells, so it is not yet clear whether activation of PR-104 by AKR1C3 can be turned to advantage. Tools for profiling reductases in human tumors (and normal tissues) will be critically important for matching bioreductive prodrugs to patients in an individualized medicine context.
2-Nitroimidazole (2-NI) hypoxia probes: can they report reductase profile as well as hypoxia?
There is much interest in the development of hypoxia biomarkers for identifying patients with the potential to benefit from hypoxia-targeted therapy. In the context of bioreductive prodrugs, the 2-NI hypoxia probes (e.g. pimonidazole, EF5) are of particular interest because their entrapment and covalent binding in cells is dependent on both hypoxia and 1eR activity. As noted above, pilot clinical data suggests that the 2-NI hypoxia probes FMISO and FAZA may identify TZP-responsive patients (1). We are exploring the potential for pairing 2-NI diagnostics with a range of bioreductive prodrugs to report both aspects of the therapeutic target (1eR activity and hypoxia) simultaneously (12,13), particularly in conjunction with 18F-EF5 PET imaging which is showing promise for hypoxic imaging in tumors (14). EF5, PR-104A and the improved TPZ analog SN30000 are all substrates for NADPH:cytochrome P450 oxidoreductase (CYPOR), a major 1eR in hypoxic cells, but whether the overlap in substrate specificity will be sufficient for diagnostic use has yet to be established. Much remains to be learned about the identity of 1eR in tumor cells, but it may ultimately be possible to profile both the 1eR and 2eR complement to select bioreductive prodrug therapy rationally.
Beyond DNA-reactive cytotoxins: Hypoxia-selective multikinase inhibitors (HS-MKI)
Given the constraints of promiscuous reductases and ubiquitous oxygen gradients in normal tissues, it is unlikely that bioreductive prodrugs can be designed to release their active effectors solely within tumors. This makes it imperative that the effectors have as much intrinsic tumor selectivity as possible. To date all bioreductive prodrugs to enter clinical development have relied on formation of DNA damage on reduction. Although the DNA replication fork targeted by these agents is arguably the most important molecular target in oncology, there is little “headspace” in conventional chemotherapy protocols for adding additional toxicity mediated by DNA damage.
In response to this need, we are designing prodrugs for hypoxia-selective release of molecularly targeted agents; the rationale is to improve therapeutic selectivity via intra-tumor activation and to selectively target signalling pathways important for hypoxic cell survival. We have recently reported a novel class of HS-MKI, exemplified by SN29966 (15,16). This quaternary ammonium salt fragments on 1e reduction of its 4-nitroimidazole trigger to release a potent irreversible pan-HER inhibitor in a hypoxia-selective manner, and provides impressive antitumor activity in xenograft models.
In conclusion, hypoxia represents an attractive target but requires a difficult balancing act from the perspective of drug design. In addition to the usual constraints of small molecule pharmacology, hypoxia-activated prodrugs need to be optimized for 1eR (with kinetics compatible with diffusion into hypoxic tissue and appropriate O2 dependence), non-substrates for 2eR, and efficiently release well-tolerated anticancer agents active against chemoresistant hypoxic cells. The clinical development of such agents will require the full toolkit of individualized cancer medicine to match compounds to tumors with the appropriate molecular and physiological features.
References
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Citation Format: William Robert Wilson. New prodrugs for targeting tumor hypoxia [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY34-01