Abstract
PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes ‘off-target’ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5–3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.
Highlights
Prodrugs have been developed that exploit oxygen depletion in human solid tumours, a pervasive feature associated with resistance to radiotherapy [1], chemotherapy [2] and immunotherapy [3,4]
To determine whether expression of aldo-keto reductase 1C3 (AKR1C3) in myeloid progenitor cells is a possible mechanism of the dose-limiting toxicity observed in humans, we first compared the aerobic sensitivity of murine and human bone marrow cells to PR-104A exposure under normoxia
This is consistent with the reports of high expression of AKR1C3 in CD34+ -positive human haematopoietic progenitor cells [26]
Summary
Prodrugs have been developed that exploit oxygen depletion (hypoxia) in human solid tumours, a pervasive feature associated with resistance to radiotherapy [1], chemotherapy [2] and immunotherapy [3,4]. Examples of hypoxia-selective prodrugs that have been evaluated clinically include tirapazamine [6], porfiromycin [7], apaziquone [8], banoxantrone [9], evofosfamide [10], tarloxotinib [11] and PR-104 [12]. PR-104 is a water-soluble phosphate pre-prodrug which undergoes systemic conversion to the di-nitro-benzamide mustard (DNBM) alcohol prodrug PR-104A in vivo [12]. One-electron reduction of PR-104A generates a nitro radical anion intermediate. Hypoxia selectivity results from rapid back oxidation of this intermediate in the presence of molecular oxygen, whilst under hypoxic conditions further reduction leads to cytotoxic metabolite formation [12]. The dominant enzyme involved in one-electron reduction of PR-104A in vitro is cytochrome
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