Mechanism of Action and Preclinical Antitumor Activity of the Novel Hypoxia-Activated DNA Cross-Linking Agent PR-104

Abstract

Purpose: Hypoxia is a characteristic of solid tumors and a potentially important therapeutic target. Here, we characterize the mechanism of action and preclinical antitumor activity of a novel hypoxia-activated prodrug, the 3,5-dinitrobenzamide nitrogen mustard PR-104, which has recently entered clinical trials. Experimental Design: Cytotoxicity in vitro was evaluated using 10 human tumor cell lines. SiHa cells were used to characterize metabolism under hypoxia, by liquid chromatography-mass spectrometry, and DNA damage by comet assay and gH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 F radiation or chemotherapy) by clonogenic assay 18 h after treatment or by tumor growth delay. Results: The phosphate ester ''pre-prodrug'' PR-104 was well tolerated in mice and converted rapidly to the corresponding prodrug PR-104A. The cytotoxicity of PR-104A was increased 10-to 100-fold by hypoxia in vitro. Reduction to the major intracellular metabolite, hydroxylamine PR-104H, resulted in DNA cross-linking selectively under hypoxia. Reaction of PR-104H with chloride ion gave lipophilic cytotoxic metabolites potentially able to provide bystander effects. In tumor excision assays, PR-104 provided greater killing of hypoxic (radioresistant) and aerobic cells in xenografts (HT29, SiHa, and H460) than tirapazamine or conventional mustards at equivalent host toxicity. PR-104 showed single-agent activity in six of eight xenograft models and greater than additive antitumor activity in combination with drugs likely to spare hypoxic cells (gemcitabine with Panc-01pancreatic tumors and docetaxel with 22RV1prostate tumors). Conclusions: PR-104 is a novel hypoxia-activated DNA cross-linking agent with marked activity against human tumor xenografts, both as monotherapy and combined with radiotherapy and chemotherapy.Hypoxia is a uniquely attractive target in oncology for two reasons. The first is that hypoxic cells are obstacles to curative cancer therapy with all major treatment modalities. Hypoxia can compromise outcomes of surgery by increasing tumor metastasis (1 -3). It is also a major cause of radioresistance because oxygen is a radiosensitizer, and multiple clinical studies have documented the importance of hypoxia determining local tumor control in radiotherapy (4 -6). Hypoxia also contributes to chemoresistance through multiple mechanisms (7), including limitations on delivery of blood-borne drugs to hypoxic regions of tumors (8,9). The second reason for targeting hypoxia is that it is a common feature of a wide variety of human tumors and is typically more severe in tumors than in normal tissues, thus providing a basis for tumor selectivity (10,11).Several strategies for exploiting tumor hypoxia are now in preclinical or clinical development (7), with the main focus on prodrugs that are activated by metabolic reduction under hypoxic conditions to form cytotoxins. Early efforts focused on quinone bioreductive drugs, such as porfiromycin (12), and 2-nitroimidazole -linked alkylating a...

Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer

“…The correlation between sensitivity to each of the DNA cross-linking agents across cell lines suggests that cellular sensitivity is dominated by DNAdamage responses that are generic across these diverse agents. SN30000 and tirapazamine also show similar cell line dependence under aerobic conditions, consistent with the idea that replication fork arrest is a common lesion across both the benzotriazine di-N-oxides (32) and cross-linking agents (27). We note that our data do not prove that compromised cross-link repair is solely responsible for the observed hypersensitivity of HR-defective cells; higher endogenous levels of DNA lesions and a correspondingly lower threshold to exogenous agents might also contribute.…”

“…Several classes of hypoxia-activated prodrugs (HAP) have been rationally developed to exploit tumor hypoxia (14). These include the clinical stage benzotriazine di-N-oxide HAP tirapazamine (25) and nitrogen mustard prodrugs TH-302 (26) and PR-104 (27), in addition to advanced preclinical compounds such as the tirapazamine analogue SN30000 (28) and a nitro-chloromethylbenzindoline (nitroCBI) that is a prodrug of a potent DNA minor groove alkylator (29). These agents are enzymatically reduced in hypoxic tumor tissue to DNA-damaging metabolites that are selectively toxic to hypoxic cells.…”

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

“…5 One such compound is PR-104, a water soluble 3,5-dinitrobenzamide mustard phosphate ester pre-prodrug that undergoes systemic hydrolysis in vivo to the corresponding alcohol prodrug PR-104A. [6][7][8] PR-104A is a bioreductive prodrug reduced under conditions of pathological hypoxia (pO 2 < 1 mmHg) 9 to the cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) derivatives, which are reactive nitrogen mustards that cause cell death through the formation of DNA inter-strand crosslinks. 6,10,11 The reduction of PR-104A under hypoxic conditions is catalyzed by members of the diflavin reductase family of proteins, especially NADPH:cytochrome P450 oxidoreductase (POR).…”

“…[6][7][8] PR-104A is a bioreductive prodrug reduced under conditions of pathological hypoxia (pO 2 < 1 mmHg) 9 to the cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) derivatives, which are reactive nitrogen mustards that cause cell death through the formation of DNA inter-strand crosslinks. 6,10,11 The reduction of PR-104A under hypoxic conditions is catalyzed by members of the diflavin reductase family of proteins, especially NADPH:cytochrome P450 oxidoreductase (POR). 12,13 However, PR-104A is also activated in an oxygen-insensitive manner by human aldo-keto reductase 1C3 (AKR1C3).…”

Prediction of Tumour Tissue Diffusion Coefficients of Hypoxia-Activated Prodrugs from Physicochemical Parameters

“…[10] At the same time the presence of more severe and extensive hypoxia in tumours than in normal tissues provides a physiological target that can potentially be exploited as a basis for tumour selectivity. [3,[11][12][13][14][15][16] Several prodrugs that are selectively activated under hypoxia are currently in clinical or preclinical development, [17][18][19][20] including the benzotriazine-di-N-oxide tirapazamine (TPZ; Fig. 1).…”