Targeting the hypoxic fraction of tumours using hypoxia-activated prodrugs
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The presence of a microenvironment within most tumours containing regions of low oxygen tension or hypoxia has profound biological and therapeutic implications. Tumour hypoxia is known to promote the development of an aggressive phenotype, resistance to both chemotherapy and radiotherapy and is strongly associated with poor clinical outcome. Paradoxically, it is recognised as a high-priority target and one of the therapeutic strategies designed to eradicate hypoxic cells in tumours is a group of compounds known collectively as hypoxia-activated prodrugs (HAPs) or bioreductive drugs. These drugs are inactive prodrugs that require enzymatic activation (typically by 1 or 2 electron oxidoreductases) to generate cytotoxic species with selectivity for hypoxic cells being determined by (1) the ability of oxygen to either reverse or inhibit the activation process and (2) the presence of elevated expression of oxidoreductases in tumours. The concepts underpinning HAP development were established over 40 years ago and have been refined over the years to produce a new generation of HAPs that are under preclinical and clinical development. The purpose of this article is to describe current progress in the development of HAPs focusing on the mechanisms of action, preclinical properties and clinical progress of leading examples.
KeywordsHypoxia-activated prodrugs TH-302 AQ4N EO9 Tirapazamine PR-104 TH-4000 Hypoxia Bioreductive drugs
The presence of hypoxia in tumours has significant biological and therapeutic implications. Biologically, hypoxia is implicated in promoting resistance to apoptosis , suppression of DNA repair pathways and promotion of genomic instability  increased invasion and metastasis , promotion of angiogenesis , modulation of tyrosine kinase-mediated cell signalling pathways , evasion from immune surveillance , induction of autophagy , hypoxia-driven changes in central metabolic pathways , global changes in the metabolome , production of L-2-hydroxyglutarate leading to altered histone methylation , metabolic adaptation to hypoxia-induced reductive stress  and the provision of a niche where cancer stem cells reside . The plethora of effects on tumour biology is mediated largely by hypoxia-inducible factors (HIF)  although HIF1-independent hypoxia responses have also been described . In terms of therapeutic implications, the seminal work conducted by Gray in the 1950s  provided the first evidence that hypoxia is an underlying cause of resistance to radiotherapy. Since then, hypoxia has been strongly implicated in resistance to several cytotoxic chemotherapy drugs and targeted therapeutics [19, 20]. Multiple mechanisms contribute to hypoxia-induced drug resistance, but as pointed out by Wilson and Hay , the generalisation that hypoxia causes resistance to all cytotoxic drugs must be viewed with caution as some drugs are effective under hypoxic conditions. This note of caution should also be extended to include targeted therapeutics following the demonstration that some (such as dasatinib) are preferentially active against cell lines in vitro under hypoxic conditions .
Whilst the extent and severity of hypoxia in tumours varies between tumour types and within individual tumours, the combined biological and therapeutic implications of hypoxia have a significant bearing on prognosis. There is now an extensive body of evidence demonstrating that hypoxia can adversely affect clinical outcome [23, 24] and this makes hypoxia a high-priority therapeutic target. The importance of hypoxia as a target has been recognised for many years, but the translation of preclinical strategies designed to target hypoxic cells into mainstream clinical use has remained stubbornly difficult to achieve. Two main approaches are being used to eradicate hypoxic cells (1) the use of ‘bioreductive drugs’ or ‘hypoxia-activated prodrugs (HAPs)’ and (2) molecularly targeted drugs aimed at exploiting biochemical responses to hypoxia, particularly HIF pathways. The current status of HIF-targeted strategies is beyond the scope of this article which focuses on HAPs, their mechanism of action and recent progress in the preclinical and clinical evaluation of leading compounds in this class of drugs. This article also describes novel approaches where HAP-based approaches are being used to improve the selectivity of targeted therapeutics.
Hypoxia-activated prodrugs (HAPs): general principles
The concept of hypoxia-activated prodrugs arose largely from the seminal work on quinone-based derivatives of Mitomycin C by Alan Sartorelli in the early 1970s . Initially, these early studies focused primarily on enzyme-activated prodrugs in a process called bioreductive activation under aerobic conditions, but this concept was extended to include hypoxia following the demonstration that Mitomycin C preferentially killed hypoxic cells in vitro . Over recent years, the term HAP has become established, but HAPs and bioreductive drugs are terms that are often used interchangeably. The principles underpinning the development of HAPs have been refined over the years, and the ‘ideal HAP’ should possess the following properties (1) ability to penetrate from a blood vessel to hypoxic cells within its pharmacokinetic lifespan; (2) preferential activation oxygen conditions that are low enough to prevent activation in normal tissues; (3) the reduced product should have the ability to kill non-proliferating cells typically found within the hypoxic fraction of tumours; and (4) the reduced product should have the ability to diffuse back into the proliferating aerobic fraction and exert a ‘bystander effect’ (Fig. 1).
Five different chemical entities have been shown to be capable of selectively targeting hypoxic cells, and these include nitro (hetero)-cyclic compounds, aromatic N-oxides, aliphatic N-oxides, quinone and transition metal complexes . Whilst these are chemically distinct classes of compounds, a modular concept for the design of HAPs has been described with the three main components being (1) a trigger, (2) a linker and (3) an effector . The effector is the cytotoxic component that is capable of killing cells within the hypoxic microenvironment, and these have typically been potent DNA-interactive agents. The purpose of the linker is to deactivate the effector, whilst the trigger group is the critical group that determines prodrug activation and hypoxia selectivity. Numerous trigger groups have been characterised, and these have to be enzymatically reduced (primarily by oxidoreductases) in order to release or activate the effector . Both one- and two-electron oxidoreductases can catalyse the reduction of the prodrug, and selectivity is determined by the ability of oxygen to reverse the activation process and/or the overexpression of oxidoreductases in tumour tissue (Fig. 1). In general terms, one-electron reduction generates a prodrug radical species that can be back-oxidised in the presence of oxygen to generate the parental prodrug and reactive oxygen species. Host defence mechanisms can detoxify these radical species, thereby reducing toxic effects in oxygenated tissue, but in the absence of oxygen, the prodrug radical species undergoes further reduction/disproportionation or fragmentation reactions to generate products that are cytotoxic  Two-electron reduction in contrast bypasses the oxygen-sensitive prodrug radical intermediate, and activation of the prodrug is typically oxygen insensitive. In this case, selectivity is largely determined by the presence of elevated levels of enzyme in tumour tissue . The mechanisms governing hypoxia-activated prodrug activation are summarised in Fig. 1, and important exceptions to these generalised mechanisms are identified here and in the main body of text below. It is also important to state that hypoxia-directed therapeutic agents are unlikely to demonstrate single-agent activity and should be used in combination with radiotherapy and/or chemotherapy that targets aerobic cells. Furthermore, in order to become effective components of combination therapies, HAPs need to be sufficiently safe with minimal side effects.
TH-302 is undergoing clinical trial, and phase I studies were published in 2011 [42, 43]. As a single agent administered intravenously, TH-302 was well tolerated. At maximum tolerated dose, adverse events included nausea, skin rash, fatigue and vomiting, and depending on the regimen used, dose-limiting toxicities were grade 3 skin and mucosal toxicities or grade 3 fatigue and vaginitis/proctitis . Two partial responses in patients with metastatic small cell lung cancer and melanoma were observed with stable disease seen in 27 out of 57 patients . A phase I study of TH-302 in combination with doxorubicin demonstrated that despite the haematologic toxicity of doxorubicin increasing when combined with TH-302, toxicities were manageable and partial responses were observed in 5 out of 15 patients with advanced soft tissue sarcoma . Furthermore, one patient with advanced melanoma participating in the phase I studies had complete resolution of Cullen’s sign together with extracranial response in lung, liver and lymph node metastasis . Promising clinical activity has also been reported in phase II trials involving TH-302 in combination with doxorubicin in advanced soft tissue sarcoma  and in combination with gemcitabine in patients with advanced pancreatic cancer . TH-302 is undergoing further phase II trials against non-small cell lung cancer (with pemetrexed, NCT02093962) and advanced melanoma (NCT01864538), and phase III clinical trials against soft tissue sarcoma (NCT01440088) and pancreatic cancer (NCT01746979, MAESTRO study) are ongoing. The results of phase III studies are eagerly anticipated.
Based on these and other favourable preclinical properties , EO9 was selected for clinical evaluation under the auspices of the New Drug Development Office in Amsterdam with phase I studies reporting partial responses in two patients with carcinomas of unknown origin and one in bile duct cancer [53, 54]. The results of phase II studies were, however, disappointing with no partial or complete responses observed. These studies concluded that EO9 had no clinical activity against NSCLC, pancreatic, breast, colorectal and gastric cancers [55, 56]. Several possible explanations were put forward to explain the poor results of these studies including the fact that EO9 was not evaluated as a classical HAP as it was only tested as a single agent. Furthermore, tumour enzymology and the presence of hypoxia in patient’s tumours were not incorporated into the design of the trials . Whilst these issues represent important deficiencies in clinical trial design, it was argued that at least some patients would have had the right ‘biochemical machinery’ to activate EO9 and other explanations for why EO9 failed must exist.
Research focused on the issue of impaired drug delivery to tumours as a possible explanation. Whilst the factors that determine drug delivery to tumours are complex, systemic pharmacokinetic profiles and the ability to extravasate and penetrate through several layers of tumour cells to reach the hypoxic fraction are two key parameters . Phase I studies had already established that EO9 was rapidly cleared from the systemic circulation following intravenous administration with half-lives of less than 20 min . This combined with experimental evidence demonstrating that EO9 does not rapidly penetrate multicell layers in vitro suggested that EO9 will not penetrate more than a few cell layers from a blood vessel within its pharmacokinetic lifespan . One method pursued to tackle this problem was to develop analogues of EO9 with better drug delivery properties, but an alternative approach designed to use EO9’s bad properties to our advantage was adopted. In the case of superficial transitional cell carcinoma of the bladder, chemotherapy is administered directly into the bladder by a catheter (intravesical administration). Intravesical administration of EO9 into the bladder would (1) circumvent the drug delivery problem observed following intravenous administration; (2) retention within the bladder for up to 1 h would extend the time EO9 was in contact with the tumour and enhance penetration; and (3) any drug that reached the systemic circulation would be rapidly cleared, thereby reducing the risk of systemic toxicity.
Following the demonstration that superficial transitional cell carcinoma of the bladder possessed the correct biochemical machinery required to activate EO9 , a new clinical trial was developed. Spectrum Pharmaceuticals sponsored the phase I study, and EO9 was administered directly into the bladder (intravesical administration) once a week for 6 weeks followed by assessment of anti-tumour efficacy 2 weeks after the final instillation. Prior to the administration of EO9, patients with multiple tumour lesions had all but one tumour surgically removed with the remaining tumour left to serve as a ‘marker lesion’ for assessing response. Complete response was defined as total ablation of the marker lesion, and eight complete responses were obtained out of a total of 12 patients entered into the study . Using an identical trial design, similar complete response rates (30 out of 45 patients) were reported in phase II studies , and recurrence-free rates at 2 years were good in comparison with other marker lesion studies [63, 64]. Following the demonstration that a single intravesical administration of EO9 within 24 h of transurethral resection was well tolerated with a good safety profile , two phase III trials commenced using this new administration schedule (NCT00598806 and NCT00461591). In April 2012, Spectrum Pharmaceuticals announced that the results of these two trials did not meet their primary endpoint of a statistically significant difference in the rate of tumour recurrence at 2 years, but analysis of the pooled data from both studies showed a statistically significant effect in favour of EO9. A further phase III study using a multi-instillation schedule (once a week for 6 weeks) has been planned (NCT01410565).
AQ4N has undergone clinical evaluation, and three phase I studies have been reported, two of which have evaluated AQ4N as a single agent [78, 79] and one in combination with radiotherapy . As a single agent, AQ4N was well tolerated up to a maximum tolerated dose of 768 mg/m2 (administered intravenously as a 30-min infusion on days 1, 8 and 15 of a 28-day cycle) with the most common adverse events being fatigue, diarrhoea, nausea, vomiting, anorexia and blue discolouration of skin and body fluids . The pharmacokinetic profile of AQ4N was dose dependent with low levels of AQ4M, and no AQ4 detected in the systemic circulation. Three patients had stable disease, two with bronchoalveolar lung cancer and ovarian cancer and the third with collecting duct renal cancer had prolonged stable disease for 25 months . In a phase I proof-of-principle pharmacodynamics study, AQ4N at a dose of 200 mg/m2 (single dose administered intravenously using a 30-min infusion) was administered 12–36 h before multiple samples of tumour and normal tissue were surgically removed from each patient. AQ4N and its metabolites were analysed by LC/MS, the distribution of AQ4 relative to blood vessels determined by confocal microscopy and the relationship between AQ4 levels and the expression of the endogenous hypoxia marker Glut-1 determined by immunohistochemistry . This study demonstrated that AQ4N was activated selectively in hypoxic regions of solid tumours and the levels of AQ4 detected exceeded those required for activity in animal models. In addition, high levels of AQ4 were detected in glioblastoma multiforme, indicating that AQ4N effectively crossed the blood–brain barrier . In combination with radiotherapy, AQ4N was well tolerated up to 447 mg/m2 administered intravenously with no dose-limiting toxicity and tumour AQ4 concentrations also exceeded levels required for activity in preclinical models. Of the eighteen patients that were assessable for response, one had a partial response, two had >50 % tumour volume reduction and nine patients had stable disease . Whether these responses were due to AQ4N or radiotherapy alone was not possible to determine but the results of this study illustrates the potential value of combination studies of AQ4N with radiotherapy. Regrettably, the clinical development of AQ4N has stalled, but new analogues of AQ4N are under development by OncoTherics.
PR-104 has activity against a range of in vivo preclinical models, and its properties mean it can target hypoxic tumours and/or the aerobic fraction of tumours expressing AKR1C3. Against T cell acute lymphoblastic leukaemia xenografts, single-agent PR-104 treatment proved more efficacious compared to a combination of vincristine, dexamethasone and l-asparaginase and activity correlated with AKR1C3 expression . This study also concludes that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from PR-104 treatment in future clinical trials. Similar studies reported complete responses in acute lymphoblastic leukaemia models and objective responses in other solid tumours, but in contrast, tumour response did not correlate with AKR1C3 levels . Other studies have demonstrated that PR-104 could be used to eradicate acute lymphoblastic leukaemia cells residing in hypoxic niches in the bone marrow . Against solid tumours, experimental and in silico modelling demonstrate that PR104/PR104A is able to penetrate into severely hypoxic regions of tumours where it is preferentially metabolised to cytotoxic metabolites . A combination of experimental and modelling techniques have also demonstrated that PR-104A can exert a bystander effect that is predicted to contribute significantly to the anti-tumour efficacy of PR-104 . PR-104 has demonstrated anti-tumour activity as a single agent against a range of solid tumour xenografts and greater than additive effects have been reported when PR-104 is used in combination with chemotherapy agents such as gemcitabine, docetaxel and sorafenib [81, 93] and/or radiotherapy . The use of pharmacological approaches to induce tumour hypoxia has also been shown to potentiate the activity of PR-104 .
PR-104 is undergoing clinical trials, and several phase I clinical trials have been completed. As a single agent, a maximum tolerated dose of 1100 mg/m2 was reported following a one dose every 21-day schedule  and 675 mg/m2 when given on a one dose per week for 3-week schedule . In both studies, PR-104 was administered by a 1-h intravenous infusion. Dose-limiting toxicities included fatigue, febrile neutropenia and infection following a once a week, every 21-day schedule and thrombocytopenia and to a lesser extent neutropenia using the more intensive schedule [95, 96]. No objective responses were reported in these studies despite the fact that PR-104A plasma AUC values exceeded the levels required for activity in preclinical models . In combination with either gemcitabine or docetaxel, severe dose-limiting myelotoxicity occurred, the impact of which was reduced by prophylactic G-CSF in the case of docetaxel . A combination of PR-104 and sorafenib in advanced hepatocellular carcinoma was also poorly tolerated with significant thrombocytopenia and neutropenia reported . PR-104A undergoes glucuronidation , and it was suggested that reduced clearance due to compromised glucuronidation in patients with advanced hepatocellular carcinoma of PR-104A was partly responsible for the toxicity observed . Recent studies in mice that do not significantly glucuronidate PR-104A confirm that the development of analogues of PR104 that are not readily glucuronidated may be able to exploit elevated AKR1C3 and/or hypoxia in hepatocellular carcinoma in humans . Based on strong preclinical data, a phase I/II study in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) has demonstrated clinical activity in 10 out of 31 patients with AML and 2 out of 10 patients with ALL . PR-104 treatment also decreased the number of hypoxic cells in the bone marrow. These positive results indicate that PR-104 is able to exploit the hypoxic niche in acute leukaemias and further clinical evaluation in this setting is warranted.
TPZ has been extensively evaluated in the clinic. Both phase I and II studies generated positive results, particularly phase II studies where TPZ was used in combination with cisplatin, etoposide and/or radiotherapy [104, 105, 106, 107]. Unfortunately, several phase III clinical trials have failed to demonstrate any survival advantage by adding TPZ to chemotherapy or radiotherapy in non-small cell lung cancer , head and neck cancer  and cervical cancer . Reasons for the failure of TPZ include failure of radiotherapy protocol compliance and lack of stratification of patients based on tumour hypoxia levels [111, 112]. Subsequent subgroup analysis of these trials using a range of endogenous markers of hypoxia proved of limited benefit in both head and neck cancers and NSCLC trials [113, 114, 115]. Whilst better methodologies for measuring hypoxia could have been employed [116, 117], the lack of a correlation between clinical response and hypoxia markers supports the clinical findings that inclusion of TPZ into combination protocols has limited if any clinical benefit. An alternative explanation for the failure of TPZ is relatively poor drug penetration into hypoxic regions of tumours. Because TPZ can be activated under comparatively mild hypoxia, it has been shown that TPZ is metabolised too rapidly to penetrate deeply into severely hypoxic tissue . Using a combination of in silico models and experimental approaches, analogues of TPZ that have better penetration and metabolism properties have been developed with SN30000 (now known as CEN-209 following licensing to Centella) emerging as a candidate for clinical development . Details of its mechanism of action have been described elsewhere , and SN30000 is likely to proceed to phase I clinical trials shortly. It is hoped that the valuable experience gained from the TPZ clinical trials is incorporated into the design of these trials .
Novel HAPs in preclinical development
Concluding remarks and future directions
One of the most pressing unmet clinical needs is the development of therapeutic agents that can eradicate the hypoxic fraction of tumour cells. Despite extensive efforts to target and kill hypoxic cells over several decades, the need for such therapeutic strategies remains a significant objective. In the field of HAPs, several compounds have made it through pre-clinical development into clinical trial, but success has so far proved elusive. These failures, however, have helped shape the development and testing of new HAPs, and there is now genuine optimism that success is imminent. Of the compounds undergoing clinical development, TH-302 is currently the ‘gold standard’ and the results of phase III trials are eagerly awaited. As described in this article, there are a number of other HAPs in clinical trial and behind these, there is a pipeline of other agents undergoing preclinical evaluation or awaiting clinical trial. Of particular interest is the development of HAP strategies designed to release targeted therapeutics (pioneered by TH-4000) within the hypoxic microenvironment of tumours, and this is an exciting development. It should also be noted that HAPs represent one approach to targeting tumour hypoxia and other areas are being actively pursued . One such avenue is tumour metabolism, and as the biology underpinning the metabolic phenotype of tumour cells and the metabolic interplay between tumour and host cells under hypoxia are unravelled, novel therapeutic targets and strategies will emerge. Despite its lack of immediate success, the field of HAP development has produced a wealth of knowledge, understanding and expertise. It is hoped that the novel approaches to targeting hypoxia under development now will take note of the principles and experience gained from over 40 years of developing HAPs and incorporate them into the design of appropriate preclinical and clinical studies.
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