Amir Ashoorzadeh

Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia

Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.

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 seco Analogues of the Duocarmycins Containing Sulfonate Leaving Groups as Hypoxia-Activated Prodrugs for Cancer Therapy

The synthesis of 19 (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate prodrugs containing sulfonate leaving groups and 7-substituted electron-withdrawing groups is reported. These were designed to undergo hypoxia-selective metabolism to form potent DNA minor groove-alkylating agents. Analogues 17 and 24, containing the benzyl sulfonate leaving group and a neutral DNA minor groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo, alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed hypoxic log(10) cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.

The effect of sulfonate leaving groups on the hypoxia-selective toxicity of nitro analogs of the duocarmycins

A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) prodrugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. They were evaluated (along with chloride leaving group analogs for comparison) for their cytotoxicity against cultures of SKOV3 and HT29 human tumor cell lines under both aerobic and hypoxic conditions. Sulfonates with neutral side chains (e.g., 5,6,7-trimethoxyindole; TMI) show consistently higher hypoxic cytotoxicity ratios (HCRs) (34-246) than the corresponding chloro analogs (2.8-3.1) in SKOV3 cells, but these trends do not hold for compounds with cationic or polar neutral side chains.

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.

Planar chiral (η6-arene)tricarbonylchromium complexes derived from mandelic acid

Abstract The enantiomers of mandelic acid have each been converted into 1,3-dioxolan-4-one derivatives. Formation of the planar chiral (η6-arene)tricarbonylchromium complexes has enabled an investigation of their potential to promote discrimination between the diastereotopic ortho and/or meta arene hydrogens during lithiation–silylation.

Abstract 5358: The hypoxia-activated EGFR-TKI TH-4000 overcomes erlotinib-resistance in preclinical NSCLC models at plasma levels achieved in a Phase 1 clinical trial

Clinical data indicate that mutant EGFR NSCLC is often heterozygous (PLoS ONE 2013; 8: e54170; PLoS ONE 2009; 4: e7464) and the presence of wild type (WT) EGFR allele is associated with limited response to EGFR-tyrosine kinase inhibitor (TKI) therapy (Cancer Sci 2008; 99:929). Tumor hypoxia upregulates WT EGFR protein and its cognate ligand TGFα via several HIF-dependent mechanisms (reviewed in: Curr Pharm Des 2013; 19:907). NSCLC is known to be a hypoxic tumor, and thus hyperactivation of WT EGFR may be an important cause of resistance to EGFR-TKI therapy. TH-4000 (formerly called PR610) is a clinical-stage hypoxia-activated prodrug that releases an irreversible EGFR-TKI under hypoxic conditions and may overcome resistance to conventional TKI therapy. We tested this hypothesis using the heterozygous WT/Δ19 EGFR PC9 tumor model and found it to be resistant to clinically relevant doses of the EGFR-TKI erlotinib; 100% of tumors progressed during treatment with human matched plasma PK exposures of erlotinib. In contrast, the homozygous Δ19 mutant EGFR tumor HCC827 was readily controlled by erlotinib (100% tumor regression). TH-4000 (15 mg/kg) produced 100% tumor regressions in both models. In vitro, PC9 cells exposed to hypoxia had elevated EGFR protein and were more resistant to erlotinib as measured by EGFR phosphorylation. In nude mice, single-dose administration of 15 mg/kg TH-4000 achieved a plasma AUC equivalent to 32 mg/m2 in human subjects, one-fifth of the maximum tolerated dose (MTD) defined in the Phase 1 trial (MTD = 150 mg/m2/week; NCT01631279). A single dose of TH-4000 (15 mg/kg) cleared rapidly from mouse plasma (T½ = 0.37 h) but had durable residency in PC9 tumors (T½ = 39 h), releasing TKI above efficacious levels for 7 days (T½β = 84 h). Consistent with these PK properties, tumor shutdown of EGFR signalling was durable, with no recovery by day 7. To confirm the mechanism of action, TH-4000 was shown to be metabolized efficiently under hypoxia using a panel of human NSCLC cell lines (rate of TKI release 0.4-2.1 nmol/hr/106 cells), a process that was inhibited by oxygen (TKI release 80% (538 vs 99 nmol/kg; p Citation Format: Adam V. Patterson, Shevan Silva, Christopher Guise, Maria Abbattista, Matthew Bull, Huai-Ling Hsu, Charles Hart, Jessica Sun, Angus Grey, Amir Ashoorzadeh, Robert Anderson, Jeff B. Smaill. The hypoxia-activated EGFR-TKI TH-4000 overcomes erlotinib-resistance in preclinical NSCLC models at plasma levels achieved in a Phase 1 clinical trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5358. doi:10.1158/1538-7445.AM2015-5358

The effect of a bromide leaving group on the properties of nitro analogs of the duocarmycins as hypoxia-activated prodrugs and phosphate pre-prodrugs for antitumor therapy

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52-286 in HT29 cells and 41-43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log(10) cell kills of 0.87-2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs.

Abstract A67: Preclinical efficacy of tarloxotinib bromide (TH-4000), a hypoxia-activated EGFR/HER2 inhibitor: rationale for clinical evaluation in EGFR mutant, T790M-negative NSCLC following progression on EGFR-TKI therapy

Tarloxotinib bromide (T) is a prodrug that releases an irreversible EGFR/HER2 inhibitor (T-TKI) under hypoxic conditions. NSCLC is known to be a hypoxic disease and wild type (WT) EGFR is upregulated by multiple hypoxia-driven mechanisms (Curr Pharm Des, 19:907). Mutant EGFR NSCLC is commonly heterozygous and may result in maintenance of WT EGFR signalling (Can Sci, 103:1946; PloS One 8:e54170). Clinical studies indicate NSCLC patients harbouring WT/mut heterozygous EGFR have significantly poorer ORR, PFS and OS on treatment with EGFR-TKI (Can Sci, 99:929). Other mechanisms of resistance to EGFR-TKI include 50-60% with T790M EGFR mutation, 8-13% with HER2 amplification, while 15-20% lack identifiable mutation/amplification events (Nat Rev Clin Onc, 11:473). The combination of cetuximab/afatinib provides an ORR of 25% and PFS of 4.6 months in T790M-negative NSCLC suggesting the persistence of HER signalling plays a role in resistance. However the high proportion of Grade 3/4 toxicity seen with cetuximab/afatinib indicates an opportunity for dose-intensification with an improved therapeutic index (Can Discov, 4:1). In addition, early clinical data on resistance to the 3rd Gen (WT EGFR-sparing) TKI rociletinib, fails to identify further mutations by NGS in some patients and describes reversion to EGFR-WT (T790) status (Can Discov, 5:713). Collectively these data support the hypothesis that WT EGFR heterozygosity may be a mechanism of resistance to current EGFR-TKI. Current EGFR-TKI lack the therapeutic index to silence WT EGFR signalling in tumors due to on-target skin/GI toxicities (Ann Oncol 18:761). Therefore we sought to examine the potency of T-TKI relative to erlotinib, afatinib and AZD9291 in five human cancer cell lines expressing WT EGFR (H1838, H2073, H1648, H125 and A431). In antiproliferative assays T-TKI was more dose-potent than erlotinib (25- to 110-fold) afatinib (4- to 32-fold) and AZD9291 (120- to 71-fold). This activity correlated with inhibition of WT EGFR phosphorylation and downstream MAPK signalling. We used a prototypic WT EGFR driven xenograft model (A431) to benchmark T activity against each EGFR-TKI by ‘retrotranslation’ of reported plasma exposure for each agent in human subjects back to the xenograft model. Only treatment with clinically relevant doses and schedules of T was associated with tumor regression and durable inhibition of WT EGFR tumor phosphorylation. Consistent with these findings, T treatment can also regress the WT EGFR NSCLC tumor models H125 and H1648, demonstrating T provides the necessary therapeutic index to inhibit WT EGFR in vivo. The transfection of WT EGFR into mutant EGFR NSCLC line PC9 (vs GFP control) conferred TGFα dependent induction of p-EGFR that was supressed by T-TKI but resistant to inhibition by erlotinib, afatinib or AZD9291. This was associated with reduced antiproliferative activity for EGFR-TKIs. Collectively these data indicate T-TKI is a dose-potent inhibitor of WT EGFR signalling and the prodrug T may possess the therapeutic index to silence WT EGFR signalling in xenograft models at plasma exposure levels achieved in a human Ph1 trial. T is under investigation in a Phase 2 clinical trial for EGFR mutant, T790M-negative, NSCLC patients who have progressed on EGFR-TKI (NCT02454842). Citation Format: Shevan Silva, Victoria Jackson, Christopher Guise, Maria Abbattista, Matthew Bull, Angus Grey, Robert Anderson, Amir Ashoorzadeh, Charles Hart, Tillman Pearce, Adam V. Patterson, Jeff B. Smaill. Preclinical efficacy of tarloxotinib bromide (TH-4000), a hypoxia-activated EGFR/HER2 inhibitor: rationale for clinical evaluation in EGFR mutant, T790M-negative NSCLC following progression on EGFR-TKI therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A67.

Abstract A66: Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS)

Tarloxotinib bromide (T), a hypoxia-activated prodrug of a potent irreversible EGFR tyrosine kinase inhibitor (T-TKI), was designed to provide clinical benefit in patients with advanced SCCHN and SCCS. In these clinical settings excessive wild type (WT) EGFR overexpression may underlie the limited activity seen with currently available EGFR-TKI where inadequate tumor signal inhibition is evident at recommended doses (Annals Oncol 2007, 18:761). In contrast to the systemic delivery of EGFR-TKI, our prodrug approach selectively targets the hypoxic microenvironment of tumors, with the goal of increasing tumor dose-intensification and thus improving the therapeutic index. Preclinical studies were conducted to test the efficacy of clinically relevant doses of T and the approved EGFR-targeted agents cetuximab and afatinib in preclinical models of SCCHN (FaDu) and SCCS (A431) that express wild type EGFR. Under varying hypoxic conditions, EGFR expression and signal transduction was assessed by western blotting (WB). Pharmacodynamic (PD) endpoints (p-EGFR, p-MAPK, p-AKT) were evaluated by WB or immunohistochemistry (IHC). Under hypoxic conditions, T was 13- and 16-fold more active than in normoxic conditions against FaDu and A431 cells, respectively, with hypoxia-specific release of T-TKI from T by LC/MS (294 and 176 pmol/h/106 cells). Notably, hypoxic exposure of FaDu cells induced a marked 25-fold induction in EGFR phosphorylation (Y1092) that was stable upon reoxygenation (≥5 h), and EGFR signalling in A431 cells was upregulated 2.3-fold post-anoxia. Dose-dependent inhibition of EGFR signalling was observed and correlated with antiproliferative activity. The SCCHN FaDu tumor xenograft had a mean hypoxic fraction (HF) of 14% as determined by IHC detection of pimonidazole binding. Treatment with 48 mg/kg (qw) T (plasma exposure = 150 mg/m2 IV in human subjects) resulted in 100% (8/8) response rate (RR). In contrast, 8 mg/kg (qw) cetuximab treatment (saturating dose) failed to control growth with 8/10 (80%) tumors progressing on treatment and 2/10 (20%) with stable disease. The SCCS A431 tumor xenograft exhibited a mean HF of 31%. Treatment with 30 mg/kg (qw) T (plasma exposure equivalent to 104 mg/m2 IV in human subjects) resulted in 100% (6/6) RR. In contrast, 5 mg/kg daily oral afatinib (plasma exposure = 33 mg daily in human subjects) failed to regress tumors with best response of stable disease in 4/6 (66%) during treatment, while 2/6 (33%) progressed during treatment. T efficacy was accompanied by marked shutdown of total tumor p-EGFR (Try1092) in both xenograft models as well as p-AKT (Ser473) and p-MAPK (Thr202/Tyr204), in the FaDu and A431 xenografts, respectively, which was not evident with the comparator treatments of cetuximab and afatinib. In preclinical models, T exhibited superior efficacy with a 100% response rate compared to cetuximab in SCCHN and afatinib in SCCS with no responses and superior pharmacodynamic effects of T on EGFR target modulation. Based on these models performed with clinically relevant plasma PK levels T may possess a superior therapeutic index relative to approved EGFR-targeted agents. These preclinical data support the weekly dose of 150 mg/m2 of T in the ongoing phase 2 clinical trial in SCCHN and SCCS (NCT02449681). Citation Format: Victoria Jackson, Shevan Silva, Maria Abbattista, Christopher Guise, Matthew Bull, Amir Ashoorzadeh, Charles Hart, Tillman Pearce, Jeff Smaill, Adam V. Patterson. Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A66.