Sophie P. Syddall

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.

Bystander or No Bystander for Gene Directed Enzyme Prodrug Therapy

Gene directed enzyme prodrug therapy (GDEPT) of cancer aims to improve the selectivity of chemotherapy by gene transfer, thus enabling target cells to convert non-toxic prodrugs to cytotoxic drugs. A zone of cell kill around gene-modified cells due to transfer of toxic metabolites, known as the bystander effect, leads to tumour regression. Here we discuss the implications of either striving for a strong bystander effect to overcome poor gene transfer, or avoiding the bystander effect to reduce potential systemic effects, with the aid of three successful GDEPT systems. This review concentrates on bystander effects and drug development with regard to these enzyme prodrug combinations, namely herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV), cytosine deaminase (CD) from bacteria or yeast with 5-fluorocytodine (5-FC), and bacterial nitroreductase (NfsB) with 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), and their respective derivatives.

Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90- to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxia-selective generation of potent DNA minor groove alkylating agents. [Mol Cancer Ther 2009;8(10):2903–13]

Pseudomonas aeruginosa NfsB and nitro-CBI-DEI – a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy

BackgroundThe nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa.FindingsInitial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect.ConclusionWe posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

Abstract B76: Cellular metabolism, murine pharmacokinetics and preclinical antitumor activity of SN29966, a novel hypoxia‐activated irreversible pan‐HER inhibitor

Hypoxia occurs in most human tumors and is associated with disease progression, resistance to conventional therapies and poor patient outcome. Hypoxia can up‐regulate HER1 by several known mechanisms, including increased mRNA translation (Franovic et al., PNAS,2007;104:13092) and delayed receptor endocytosis (Wang et al., Nat Med.,2009;15:319). We have developed SN29966, a hypoxia‐activated prodrug of the irreversible pan‐HER inhibitor SN29926, to target and exploit hypoxic cells and thereby broaden the therapeutic index of this class of agent. Quaternization of the aminebearing Michael acceptor masks activity SN29926, and one‐electron reduction provides selective release under hypoxia. This prodrug design confers 62‐fold deactivation of inhibitor activity with respect to HER1 autophosphorylation in A431 cells (Smaill et al., this meeting). In a panel of HER1/2 expressing cell lines SN29966 showed hypoxia‐dependent inhibition of proliferation (hypoxic/oxic IC50 ratios of 63, 38, 31 and 19 in BT474, A431, SKOV3 and SKBR3 cells, respectively), a property SN29926 lacked (IC50 ratios 0.7–1.1). SN29926 was generated from SN29966 under hypoxic conditions at a rate of 200–500 pmol/hr/106 cells. Oxic production was ∼1 pmol/hr/106 cells. Plasma and A431 tumor pharmacokinetics (PK) of prodrug SN29966 and inhibitor were measured in nude mice by LC/MS/MS detection (with D6 internal standards) following administration at their respective MTDs (133 and 75 umol/kg; ip). Prodrug SN29966 gave a plasma AUC0‐72h of 2016 umol‐h/L, some ∼110‐fold greater than achieved for administration of inhibitor SN29926 (18 umol‐h/L). The latter gave a tumor AUC0‐inf of 100 umol‐h/kg with a half‐life (t½) of 9 h. In contrast the prodrug SN29966 gave a tumor AUC0‐72h of 2245 umol‐h/kg with a stable tumor tissue concentration of ∼ 30 umol/kg out to 72 h, such that a t½ could not be determined. Consistent with this long prodrug residency, SN29926 released from prodrug had a t½ in tumor tissue of >72h, providing an AUC0‐72h of 464 umol‐h/kg. Thus the AUC of SN29926 in A431 tumors was at least 4.6‐fold higher after administration of prodrug SN29966 than following administration of inhibitor SN29926 itself at equivalent toxicity. In A431 tumor growth delay studies, SN29966 (113 umol/kg, q4dx6, ip) induced tumor regressions (12/12) with no recovery of growth by day 36 (tumor volume 44 ±19 mm3), whereas controls grew rapidly (day 8 tumor volume 1043 ±109 mm3). Comparative administration of inhibitor SN29926 (63 umol/kg, q4dx6, ip) provided tumor stasis, but with rapid growth following cessation of treatment (day 36 tumor volume 642 ±191 mm3), a difference that was significant from prodrug (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B76.

Abstract A247: Mechanism of action of the hypoxia-activated irreversible pan-HER inhibitor SN29966.

Hypoxia occurs in most human tumors and is associated with disease progression, treatment resistance and poor patient outcome. We have developed the hypoxia-activated prodrug SN29966, designed to release the irreversible pan-HER inhibitor SN29926, following one-electron reduction by hypoxic cells (Smaill et al, Mol Cancer Ther., 2009; 8(12 Suppl), C46). Pharmacokinetic (PK) studies in nude mice bearing A431 tumor xenografts indicated SN29966 has a long tumor half-life (>3 days) and releases SN29926 in tumors. SN29966 demonstrated single agent activity in nude mice bearing A431 and SKOV3 xenografts, inducing striking tumor regressions in both models (Patterson et al, Mol Cancer Ther., 2009; 8(12 Suppl), B76). PR509 and PR610, clinical candidates developed from SN29966, are currently undergoing comparative evaluation with Phase I trials anticipated in early 2012. The single-agent antitumor activity of SN29966 is arguably counter-intuitive given that it is designed to target hypoxic cells within tumors. This activity may arise from a number of contributing mechanisms including; (i) bioactivity of the unreduced prodrug; (ii) local redistribution of released inhibitor in the tumor; (iii) liver metabolism and circulating inhibitor and (iv) a long tumor half-life allowing for targeting of both chronic and cycling hypoxia. To critically assess the relative contribution of each to the mechanism of action of SN29966 we performed a number of studies. We prepared SN31950, a prodrug of SN29926 designed to be incapable of one-electron fragmentation. In target modulation and anti-proliferative assays SN31950 showed no hypoxia-dependent activity. The murine A431 tumor PK of SN29966 and SN31950 demonstrated that at an equimolar dose (20 μmol/kg, ip), both prodrugs gave comparable tumor exposures (AUC0–72h: SN31950, 50 μmol*h/kg; SN29966, 57 μmol*h/kg). In contrast, the tumor exposure of SN29926 released from each prodrug differed by 40-fold (AUC0–72h: SN29926 from SN31950, 0.3 μmol*h/kg; SN29926 from SN29966, 12 μmol*h/kg). Plasma exposure of each prodrug was comparable, as were levels of SN29926 in plasma (presumed mainly due to hepatic prodrug metabolism). Consistent with the observed lack of inhibitor release in A431 tumors, SN31950 was inactive against A431 tumors in growth delay assays. To confirm the hypoxia-dependent nature of SN29966 inhibitor release in A431 tumors we re-oxygenated tumors in mice breathing 100% oxygen at 2.5 atm in a hyperbaric chamber. Accordingly, mice showed a marked reduction (56%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A247.

Abstract C46: Design and identification of the novel hypoxia‐activated irreversible pan‐HER inhibitor SN29966

Several irreversible pan‐HER inhibitors (HKI‐272, BIBW‐2992, PF299) are under development and demonstrate encouraging activity against erlotinib resistant non‐small cell lung cancer expressing mutant forms of HER1. However, dose limiting toxicities mirror that of erlotinib and are attributed to inhibition of HER1 in gastrointestinal and skin tissues. To introduce additional tumor selectivity to irreversible pan‐HER inhibitors and thereby broaden their therapeutic index, we have sought to utilise tumor hypoxia as a physiological target that supports tumor‐selective bioreduction. To achieve this we have developed a series of hypoxia‐activated prodrugs of the known irreversible pan‐HER inhibitor SN29926. Five nitromethylaryl quaternary ammonium bromide (NMQ) prodrugs were synthesised by quaternizing the tertiary amine of SN29926 with five nitroheterocyclic alpha‐methyl bromides. Three further quaternary salts were prepared as Chemical‐Biology Tools (CBTs) designed as controls to probe aspects of the mechanism of action of the NMQ prodrugs. The NMQ prodrugs and CBTs were compared, relative to SN29926, in a range of assays to identify a lead compound that (i) is deactivated under oxic conditions with respect to A431 cellular HER1 target modulation and proliferation, (ii) efficiently fragments following one‐electron reduction in an oxygen inhibited manner to release the irreversible pan‐HER inhibitor, (iii) displays increased anti‐proliferative activity against A431 cells under hypoxia. All of the NMQ/CBT prodrugs were successfully deactivated with respect to inhibition of A431 HER1 autophosphorylation (62‐ to 201‐fold) and proliferation under oxic conditions (12‐ to 294‐fold). Pulse and steady‐state radiolysis under nitrogen determined their one‐electron reduction potentials (−388 to −493 mV) and demonstrated that only two of the NMQ prodrugs (SN29965, SN29966) efficiently fragment following one‐electron reduction (first‐order rate constants of 90 and 130 s−1, respectively). SN29966 alone demonstrated significant anti‐proliferative activity against A431 cells under hypoxia (hypoxic/oxic IC50 ratio 38). Preliminary growth delay screening of SN29966 and the CBTs in advanced A431 xenografts (∼600 mm3; hypoxic fraction of 32% ± 13%) demonstrated that the CBTs lacked efficacy, while SN29966 induced tumor regressions, with greater than 30 days tumor control using a well tolerated three‐dose schedule (133 umol/kg/dose, q4dx3). In summary, prodrug SN29966 is deactivated relative to the parent inhibitor, efficiently fragments following one‐electron reduction, is selective against hypoxic A431 cells and has remarkable single‐agent activity against hypoxic A431 xenografts using a conservative treatment schedule. We therefore identify SN29966 as a “first‐in‐class” hypoxia‐activated irreversible pan‐HER inhibitor that has significant clinical potential. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C46.

Abstract B88: Discovery, characterization, and engineering of bacterial nitroreductases for gene-directed enzyme prodrug therapy.

Tumor-targeting viruses and bacteria hold great promise as anti-cancer agents. They kill cells by entirely different mechanisms to radio- and chemotherapies, and have potential to synergize with these treatments without overlapping toxicities. Furthermore, these agents can be ‘armed’ with genes that encode enzymes that activate prodrugs - compounds that are deactivated in their administered form, but become highly toxic upon metabolic activation. This not only improves killing of infected cells, but also neighboring non-infected cells, as the prodrug metabolites can diffuse locally and exert a bystander effect. A highly efficient activating enzyme in partnership with a prodrug that has a strong bystander effect can address some of the historical limitations of cancer gene therapy including the inability of biological vectors to reach every target cell. Phase I/II trials of the first-generation nitroaromatic prodrug CB1954 in conjunction with the prototype gene therapy nitroreductase, Escherichia coli NfsB, have been conducted in prostate and liver cancer. These trials provided some evidence of anti-tumor activity but, due to dose-limiting hepatotoxicity, the highest achievable plasma concentration of CB1954 was less than 1% of NfsB9s K m . As well as highlighting a need for more efficient nitroreductase enzymes, this has fuelled a search for superior nitroaromatic prodrugs. The next-generation dinitrobenzamide mustard prodrug PR-104A is not only 5–50 fold more dose-potent upon activation, but also better tolerated in humans (MTD 1100 mg/m 2 vs 24 mg/m 2 ; q3w, iv). However, E. coli NfsB also has relatively poor (millimolar) affinity for this substrate. To discover more efficient nitroreductases we developed screens for genotoxic prodrug activation, based on their ability to induce reporter genes linked to the E. coli SOS (DNA damage repair) response. We used these to screen a large library of candidate enzymes for DNBM activity, and selected E. coli NfsA as a top candidate for further improvement by random and targeted mutagenesis. High throughput screening of large error prone PCR libraries coupled with medium throughput screening of targeted mutagenesis libraries revealed 10 individual mutations that significantly increased NfsA activity. These mutations were then combined in a synthetic “smart” library, from which eight poly-mutant enzymes were selected for kinetic analysis. Relative to wild type the engineered variants display an 18–40 fold improvement in PR-104A K m with respect to E. coli NfsA, and are 860–1700 fold better than NfsB. Importantly they also retain, or are improved in, their ability to co-metabolize preferred 2-nitroimidazole probes with PET-imaging capabilities (see abstract: Patterson et al, “Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5”). The enhanced prodrug activation and in vivo imaging potential of these enzymes is now being evaluated in human gene therapy models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B88.