Susan M. Pullen

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

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 γH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 ± 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-01 pancreatic tumors and docetaxel with 22RV1 prostate 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.

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

DNA Cross-Links in Human Tumor Cells Exposed to the Prodrug PR-104A: Relationships to Hypoxia, Bioreductive Metabolism, and Cytotoxicity

PR-104, currently in clinical trial, is converted systemically to the dinitrobenzamide nitrogen mustard prodrug PR-104A, which is reduced selectively in hypoxic cells to cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) metabolites. Here, we evaluate the roles of this reductive metabolism, and DNA interstrand cross-links (ICL), in the hypoxic and aerobic cytotoxicity of PR-104. Using a panel of 9 human tumor cell lines, cytotoxicity was determined by clonogenic assay after a 2-hour aerobic or hypoxic exposure to PR-104A. PR-104H and PR-104M were determined by high performance liquid chromatography/mass spectrometry, and ICL with the alkaline comet assay. Under hypoxia, the relationship between ICL and cell killing was similar between cell lines. Under aerobic conditions, there was a similar relationship between ICL and cytotoxicity, except in lines with very low rates of aerobic reduction of PR-104A (A2780, C33A, H1299), which showed an ICL-independent mechanism of PR-104A cytotoxicity. Despite this, in xenografts from the same lines, the frequency of PR-104-induced ICL correlated with clonogenic cell killing (r(2) = 0.747) with greatest activity in the fast aerobic metabolizers. In addition, changing levels of hypoxia in SiHa tumors modified both ICL frequency and tumor growth delay in parallel. We conclude that both aerobic and hypoxic nitroreduction of PR-104A contribute to the monotherapy antitumor activity of PR-104 in human tumor xenografts, and that ICL are responsible for its antitumor activity and represent a broadly applicable biomarker for tumor cell killing by this novel prodrug.

Bystander Effects of Bioreductive Drugs: Potential for Exploiting Pathological Tumor Hypoxia with Dinitrobenzamide Mustards

Abstract Wilson, W. R., Hicks, K. O., Pullen, S. M., Ferry, D. M., Helsby, N. A. and Patterson, A. V. Bystander Effects of Bioreductive Drugs: Potential for Exploiting Pathological Tumor Hypoxia with Dinitrobenzamide Mustards. Radiat. Res. 167, 625–636 (2007). Tumor hypoxia is an important therapeutic target, and it can potentially be exploited by hypoxia-activated prodrugs. However, physiological hypoxia in normal tissues is a limitation. One solution would be to confine activation to severely (pathologically) hypoxic tissue, using hypoxia-activated prodrugs that provide a bystander effect through diffusion of the activated cytotoxin to adjacent regions at intermediate oxygen concentrations (associated with partial radioresistance). To evaluate this requirement, we identified five hypoxia-activated prodrugs with at least 10-fold higher potency against a cell line (A549-P540puro) overexpressing human cytochrome P450 reductase (P450R) relative to A549-Lo21 cells with 200-fold lower P450R activity. Bystander killing by these hypoxia-activated prodrugs was tested in anoxic multicellular layer co-cultures of these two cell lines. Cytotoxic potency against A549-Lo21 cells was unaffected by the presence of A549-P450puro cells for tirapazamine and RSU-1069 but increased more than 10-fold for the aziridinyldintrobenzamide CB 1954, more than 14-fold for the corresponding nitrogen mustard SN 23862, and 15-fold for its water-soluble analog SN 23816. The cytotoxic extracellular metabolites resulting from hypoxic nitroreduction of CB 1954 and SN 23862 by A549-P450puro cells were identified by LC/MS and bioassay methods. For SN 23862, these included the 2-amine metabolite, previously, identified as the bystander metabolite from aerobic activation by the E. coli nfsB nitroreductase, but also novel di-reduced metabolites. Cytotoxicity of SN 23862 to A549-P450puro cells was inhibited by lower concentrations of oxygen than for tirapazamine. The combination of selective activation under severe hypoxia with an efficient bystander effect identifies the dinitrobenzamide mustards for further development as hypoxia-activated prodrugs.

Comparison of aromatic and tertiary amine N-oxides of acridine DNA intercalators as bioreductive drugs: Cytotoxicity, DNA binding, cellular uptake, and metabolism

Some N-oxide derivatives of DNA intercalators are bioreductive prodrugs that are selectively toxic under hypoxic conditions. The hypoxic selectivity is considered to result from an increase in DNA binding affinity when the N-oxide moiety is reduced. This study investigated whether differences in DNA binding affinity between N-oxides and their corresponding amines, measured by equilibrium dialysis, can account for the hypoxic cytotoxicity ratios (HCR) of tertiary amine N-oxide (-tO) and aromatic N-oxide (-aO) derivatives of the 1-nitroacridine nitracrine (NC) and its non-nitro analogue 9-[3-(N,N-dimethylamino)propylamino]acridine (DAPA). Cytotoxicity was measured in aerobic and hypoxic suspensions of Chinese hamster ovary (CHO) AA8 cells by clonogenic assay. HCR were much greater for NC-tO (820-fold) than for NC (5-fold) or NC-aO (4-fold), whereas DAPA and its N-oxides lacked hypoxic selectivity (1-fold). DNA binding measurements demonstrated that binding affinity is lowered more by aromatic than tertiary amine (side-chain) N-oxides, an observation that does not correlate with HCR. Compounds were accumulated in cells to high concentrations (C(i)/C(e) approximately 10-200), with the exception of the tertiary amine N-oxides, for which the ratio of intracellular to extracellular drug was less than unity. For NC-tO this probably resulted from low pK(a) values for both the acridine chromophore and the side-chain, whereas DAPA-tO may be too hydrophilic for efficient membrane permeation. Bioreductive drug metabolism, assessed by HPLC, was faster for the NC than the DAPA N-oxides. The high HCR of NC-tO relative to NC-aO is ascribed to the rapid and selective reduction of its N-oxide moiety, followed by activation of the NC intermediate by O(2)-sensitive reduction of its 1-nitro group to the corresponding 1-amine. The metabolism studies suggest that unmasking of DNA binding affinity by reductive removal of the N-oxide moiety, although not the only determinant, is important and needs to occur before nitroreduction for optimal effect.

Nitrobenzyl mustard quaternary salts: A new class of hypoxia-selective cytotoxins capable of releasing diffusible cytotoxins on bioreduction

PURPOSE To explore the utility of a new class of compounds, nitrobenzyl mustard quaternary salts, as hypoxia-selective prodrugs of diffusible cytotoxins. METHODS AND MATERIALS The parent compound N,N-bis(2-chloroethyl)-N-methyl-N-(2-nitrobenzyl)ammonium chloride (SN 25246) was prepared by reaction of 2-nitrobenzyl chloride with N-methyldiethanolamine, and reaction of the resulting quaternary diol with thionyl chloride at room temperature. The rate of release of mechlorethamine from this compound in the presence of cells under aerobic and hypoxic conditions was determined by trapping with diethyldithiocarbamate. Cytotoxicity was assessed by clonogenic assay of stirred suspension cultures of EMT6 cells, and also in intact and dissociated EMT6 spheroids. In vivo activity was evaluated in mice bearing SC KHT tumors. RESULTS The parent compound is a stable, water-soluble compound with an E(1) of -369 mV. It releases mechlorethamine, and shows selective toxicity towards hypoxic EMT6 cells in culture, increasing with time to several 1000-fold after 4 h. It is much more active against intact than dissociated EMT6 spheroids, and shows low but statistically significant activity against KHT tumors in vivo. CONCLUSION Nitrobenzyl mustard quaternary salts are a new class of hypoxia-selective cytotoxin. The parent compound shows very high selectivity for hypoxic cells in vitro, and undergoes reduction in hypoxic cells, releasing mechlorethamine which can back-diffuse to kill surrounding oxygenated cells.

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

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simultaneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapazamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemotherapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppression of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2 were knocked down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies. Mol Cancer Ther; 13(11); 2501–14. ©2014 AACR.

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

In Vitro and In Vivo Models for Evaluation of GDEPT

The vectors currently available for gene therapy of cancer rarely achieve expression of therapeutic genes in more than a small fraction of the cells in solid tumors. This makes therapeutic success critically dependent on secondary events, known as bystander effects, by which transgene expression leads to the death of nontransduced tumor cells. An efficient bystander effect has the potential to compensate for spatially nonuniform expression of therapeutic genes, and its optimization is therefore an important goal in gene therapy of cancer. Here, we describe protocols for quantifying bystander effects using in vitro and in vivo experimental models. The generation of efficient bystander killing is the key rationale for gene-dependent enzyme–prodrug therapy (GDEPT), in which a suicide gene codes for a prodrug-activating enzyme (PAE) that generates a diffusible cytotoxin. In a well-studied example, the prodrug ganciclovir (GCV) is activated by herpes simplex virus-thymidine kinase (HSV-TK) to form a phosphorylated metabolite that diffuses between cells via gap junctions (1,2). In other cases, the bystander metabolite can diffuse across cell membranes, as for 5-fluorouracil (5-FU) generated from 5-fluorocytosine (5-FC) by bacterial or yeast cytosine deaminase (CD) (3,4). Several newer GDEPT systems generate freely diffusible (membrane-permeable) alkylating metabolites with the added attractive feature of killing noncycling as well as cycling tumor cells; these include oxidation of cyclophosphamide and other oxazaphosphorines by cytochrome P450 2B1/cytochrome P450 reductase (5), hydrolysis of glutamate derivatives of aromatic nitrogen mustards (prodrugs CMDA and ZD2767P) by Pseudomonas carboxypeptidase G2 (6,7), and reduction of the dinitrobenzamide CB 1954 (see Fig. 1) to its 4-hydroxylamine by the Escherichia coli aerobic nitroreductase NTR (8–10). The CB 1954/NTR

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

PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.