Michael P. Hay

Targeting hypoxia in cancer therapy

Hypoxia is a feature of most tumours, albeit with variable incidence and severity within a given patient population. It is a negative prognostic and predictive factor owing to its multiple contributions to chemoresistance, radioresistance, angiogenesis, vasculogenesis, invasiveness, metastasis, resistance to cell death, altered metabolism and genomic instability. Given its central role in tumour progression and resistance to therapy, tumour hypoxia might well be considered the best validated target that has yet to be exploited in oncology. However, despite an explosion of information on hypoxia, there are still major questions to be addressed if the long-standing goal of exploiting tumour hypoxia is to be realized. Here, we review the two main approaches, namely bioreductive prodrugs and inhibitors of molecular targets upon which hypoxic cell survival depends. We address the particular challenges and opportunities these overlapping strategies present, and discuss the central importance of emerging diagnostic tools for patient stratification in targeting hypoxia.

A molecule targeting VHL-deficient renal cell carcinoma that induces autophagy.

Renal cell carcinomas (RCCs) are refractory to standard therapies. The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in 75% of RCCs. By screening for small molecules selectively targeting VHL-deficient RCC cells, we identified STF-62247. STF-62247 induces cytotoxicity and reduces tumor growth of VHL-deficient RCC cells compared to genetically matched cells with wild-type VHL. STF-62247-stimulated toxicity occurs in a HIF-independent manner through autophagy. Reduction of protein levels of essential autophagy pathway components reduces sensitivity of VHL-deficient cells to STF-62247. Using a yeast deletion pool, we show that loss of proteins involved in Golgi trafficking increases killing by STF-62247. Thus, we have found a small molecule that selectively induces cell death in VHL-deficient cells, representing a paradigm shift for targeted therapy.

Structure−Activity Relationships of 1,2,4-Benzotriazine 1,4-Dioxides as Hypoxia-Selective Analogues of Tirapazamine

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides (BTO) to examine structure-activity relationships (SAR) for ring substitution. The electronic, hydrophobic, and steric parameters of substituents at the 5-, 6-, 7-, and 8-positions were systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)] of the analogues were determined. For each compound, we determined cell killing of mouse SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio (HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation assay, the data from which correlated well with that derived by the clonogenic endpoint. Most substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogues less soluble than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of -456 mV) and correlated well with the electronic parameter sigma for substituents at the 5-, 6-, 7-, and 8-positions. Aerobic cytotoxic potency showed a strong positive correlation with E(1) (i.e., electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 to -400 mV. Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-donating substituents) with the exception of the 8-CF(3) analogue, which had an HCR of 112 against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents in BTO analogues can be used to predictably vary one-electron reduction potentials and also provide a much better definition than previously of the optimum range of these reduction potentials for a desirable biological activity profile (high HCR, RHT, and solubility).

Pharmacokinetic/Pharmacodynamic Modeling Identifies SN30000 and SN29751 as Tirapazamine Analogues with Improved Tissue Penetration and Hypoxic Cell Killing in Tumors

Purpose: Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but has limited clinical activity, in part because of poor extravascular penetration. Here, we identify improved TPZ analogues by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization. Experimental design: The SR-PKPD model was used to guide the progression of 281 TPZ analogues through a hierarchical screen. For compounds exceeding hypoxic selectivity thresholds in single-cell cultures, SR-PKPD model parameters (kinetics of bioreductive metabolism, clonogenic cell killing potency, diffusion coefficients in multicellular layers, and plasma pharmacokinetics at well tolerated doses in mice) were measured to prioritize testing in xenograft models in combination with radiation. Results: SR-PKPD–guided lead optimization identified SN29751 and SN30000 as the most promising hypoxic cytotoxins from two different structural subseries. Both were reduced to the corresponding 1-oxide selectively under hypoxia by HT29 cells, with an oxygen dependence quantitatively similar to that of TPZ. SN30000, in particular, showed higher hypoxic potency and selectivity than TPZ in tumor cell cultures and faster diffusion through HT29 and SiHa multicellular layers. Both compounds also provided superior plasma PK in mice and rats at equivalent toxicity. In agreement with SR-PKPD predictions, both were more active than TPZ with single dose or fractionated radiation against multiple human tumor xenografts. Conclusions: SN30000 and SN29751 are improved TPZ analogues with potential for targeting tumor hypoxia in humans. Novel SR-PKPD modeling approaches can be used for lead optimization during anticancer drug development. Clin Cancer Res; 16(20); 4946–57. ©2010 AACR.

A 2-nitroimidazole carbamate prodrug of 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H -benz[e]indole (amino-SECO-CBI-TMI) for use with ADEPT and GDEPT

The synthesis of a 2-nitroimidazol-5-ylmethyl carbamate prodrug 10 of the potent minor groove alkylating agent amino-seco-CBI-TMI 3 is described. Chemical, radiolytic, and enzymic reductions of a model 2-nitroimidazol-5-yl carbamate 8 show release of the amine effector upon reduction. Prodrug 10 gives a ten fold increase in cytotoxicity against human ovarian carcinoma SKOV3 cells in the presence of E. coli B nitroreductase (NTR) and a 21-fold increase in cytotoxicity against a SKOV3 cell line (SC3.2) transfected with the gene for NTR. The cytotoxicity of 10 increased 15- to 40-fold under hypoxia. Prodrug 10 has significant potential as a prodrug for use in ADEPT and GDEPT applications, and as a hypoxia-selective cytotoxin.

The 2-nitroimidazole EF5 is a biomarker for oxidoreductases that activate the bioreductive prodrug CEN-209 under hypoxia

Purpose: Benzotriazine-N-oxide bioreductive prodrugs such as tirapazamine and its improved analogue CEN-209 (SN30000) have potential for exploiting hypoxia in tumors. Here, we test the hypothesis that the 2-nitroimidazole EF5, in clinical development for both immunohistochemical and positron emission tomography imaging of hypoxia, can detect not only hypoxia but also the one-electron reductases required for activation of these hypoxia-targeted prodrugs. Experimental Design: Aerobic and hypoxic covalent binding of [14C]-EF5 was determined in human tumor cell lines, including lines with overexpression of NADPH:cytochrome P450 oxidoreductase (CYPOR), and reductive metabolism of tirapazamine and CEN-209 by mass spectrometry. DNA damage response was measured by γH2AX formation. Bioreductive metabolism was modulated in HCT116 tumor xenografts by overexpression of CYPOR and breathing of hyperbaric oxygen or 10% oxygen. Results: Overexpression of CYPOR induced similar 2- to 4-fold increases in EF5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines, and similar enhancement of γH2AX formation. EF5 binding and metabolic reduction of the prodrugs were highly correlated in a panel of 14 hypoxic tumor cell lines. In HCT116 xenografts, CYPOR overexpression also significantly increased EF5 binding and CEN-209 reduction, and modification of tumor hypoxia caused similar changes to the bioreductive activation of both agents, resulting in a strong correlation between EF5 binding and CEN209-induced DNA damage (R2 = 0.68, P < 0.0001) at the individual tumor level. Conclusions: EF5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs because of its potential for interrogating reductase activity as well as hypoxia in individual tumors. Clin Cancer Res; 18(6); 1684–95. ©2011 AACR.

Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.

Selective Potentiation of the Hypoxic Cytotoxicity of Tirapazamine by Its 1-N-Oxide Metabolite SR 4317

Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2–3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 × 10−6 cm2s−1, with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.

Homologous recombination repair-dependent cytotoxicity of the benzotriazine di-N-oxide CEN-209: Comparison with other hypoxia-activated prodrugs

CEN-209 (SN30000) is a second-generation benzotriazine di-N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells (Rad51d⁻/⁻), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di-N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H₂O₂ production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di-N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.

A novel enediyne prodrug for antibody-directed enzyme prodrug therapy (ADEPT) using E. coli B nitroreductase

Abstract The synthesis of a novel enediyne 2 , and its cytotoxicity and activation by the nitroreductase enzyme NR2 from Esherichia coli B, are described. In contrast to closely related analogues, 2 exhibits a 90-fold increase in cytotoxicity against UV4 cells in the presence of the enzyme and NADH, suggesting its potential as a prodrug for Antibody-Directed Enzyme Prodrug Therapy in conjunction with E. coli nitroreductase.