Design, Synthesis and Anticancer Evaluation of Nitroimidazole Radiosensitisers.

The role of hypoxic tumour cells in resistance to radiotherapy, and in suppression of immune response, continues to endorse tumour hypoxia as a bona fide, yet largely untapped, drug target. Radiotherapy innovations such as stereotactic body radiotherapy herald new opportunities for classical oxygen-mimetic radiosensitisers. Only nimorazole is used clinically as a radiosensitiser, and there is a dearth of new radiosensitisers in development.

Tissue Pharmacokinetic Properties and Bystander Potential of Hypoxia-Activated Prodrug CP-506 by Agent-Based Modelling.

Hypoxia-activated prodrugs are bioactivated in oxygen-deficient tumour regions and represent a novel strategy to exploit this pharmacological sanctuary for therapeutic gain. The approach relies on the selective metabolism of the prodrug under pathological hypoxia to generate active metabolites with the potential to diffuse throughout the tumour microenvironment and potentiate cell killing by means of a "bystander effect". In the present study, we investigate the pharmacological properties of the nitrogen mustard prodrug CP-506 in tumour tissues using spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling.

Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes 'off-target' two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues.

Radiosensitisation of SCCVII tumours and normal tissues in mice by the DNA-dependent protein kinase inhibitor AZD7648.

Background And Purpose: Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice.

Materials And Methods: Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay.

Selectively Targeting Tumor Hypoxia With the Hypoxia-Activated Prodrug CP-506.

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

Purpose: Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue.

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear.

Benzotriazine Di-Oxide Prodrugs for Exploiting Hypoxia and Low Extracellular pH in Tumors.

Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments.

Schedule-dependent potentiation of chemotherapy drugs by the hypoxia-activated prodrug SN30000.

Hypoxia-activated prodrugs (HAPs) are hypothesized to improve the therapeutic index of chemotherapy drugs that are ineffective against tumor cells in hypoxic microenvironments. SN30000 (CEN-209) is a benzotriazine di--oxide HAP that potentiates radiotherapy in preclinical models, but its combination with chemotherapy has not been explored. Here we apply multiple models (monolayers, multicellular spheroids and tumor xenografts) to identify promising SN30000/chemotherapy combinations (with chemotherapy drugs before, during or after SN30000 exposure).

An Intratumor Pharmacokinetic/Pharmacodynamic Model for the Hypoxia-Activated Prodrug Evofosfamide (TH-302): Monotherapy Activity is Not Dependent on a Bystander Effect.

Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking.

An agent-based model for drug-radiation interactions in the tumour microenvironment: Hypoxia-activated prodrug SN30000 in multicellular tumour spheroids.

Multicellular tumour spheroids capture many characteristics of human tumour microenvironments, including hypoxia, and represent an experimentally tractable in vitro model for studying interactions between radiotherapy and anticancer drugs. However, interpreting spheroid data is challenging because of limited ability to observe cell fate within spheroids dynamically. To overcome this limitation, we have developed a hybrid continuum/agent-based model (ABM) for HCT116 tumour spheroids, parameterised using experimental models (monolayers and multilayers) in which reaction and diffusion can be measured directly.

Bystander Effects of Hypoxia-Activated Prodrugs: Agent-Based Modeling Using Three Dimensional Cell Cultures.

Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking.

Evofosfamide for the treatment of human papillomavirus-negative head and neck squamous cell carcinoma.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models.

Next-Generation Hypoxic Cell Radiosensitizers: Nitroimidazole Alkylsulfonamides.

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6-indeno[5,6-][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice.

Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice.

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of HO. Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1).

Efficient Protocol for the Identification of Hypoxic Cell Radiosensitisers.

An evolution in radiotherapy practice is leading to greater use of stereotactic body radiotherapy (SBRT), raising the prospect of increased hypoxic cell radioresistance. New clinical interest in nitroimidazole radiosensitisers, combined with appropriate biomarkers, signals a revival for radiosensitisers in the context of SBRT. Our interest in modifiers of radiation therapy led us to revisit this area and we have identified a new class of nitroimidazole radiosensitiser.

Pharmacokinetic modeling of ascorbate diffusion through normal and tumor tissue.

Ascorbate is delivered to cells via the vasculature, but its ability to penetrate into tissues remote from blood vessels is unknown. This is particularly relevant to solid tumors, which often contain regions with dysfunctional vasculature, with impaired oxygen and nutrient delivery, resulting in upregulation of the hypoxic response and also the likely depletion of essential plasma-derived biomolecules, such as ascorbate. In this study, we have utilized a well-established multicell-layered, three-dimensional pharmacokinetic model to measure ascorbate diffusion and transport parameters through dense tissue in vitro.

Photodegradation of the benzotriazine 1,4-Di-N-oxide hypoxia-activated prodrug SN30000 in aqueous solution.

SN30000 is a benzotriazine di-N-oxide which is selectively toxic to radio-resistant hypoxic cells in tumours. Given the complex photochemistry of some aromatic N-oxides, we evaluated the potential for photodegradation of SN30000 solutions. Initial studies demonstrated significant oxygen-insensitive degradation under normal laboratory lighting conditions.

Novel nitroimidazole alkylsulfonamides as hypoxic cell radiosensitisers.

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selective cytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemical properties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroimidazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with charged amine groups contributing to increased electron affinity.

Design of optimized hypoxia-activated prodrugs using pharmacokinetic/pharmacodynamic modeling.

Hypoxia contributes to resistance of tumors to some cytotoxic drugs and to radiotherapy, but can in principle be exploited with hypoxia-activated prodrugs (HAP). HAP in clinical development fall into two broad groups. Class I HAP (like the benzotriazine N-oxides tirapazamine and SN30000), are activated under relatively mild hypoxia.

Hypoxia-directed drug strategies to target the tumor microenvironment.

Hypoxia is an important component of the tumor microenvironment and has been the target of drug discovery efforts for almost half a century. These efforts have evolved from offsetting the impact of hypoxia on radiotherapy with oxygen-mimetic radiosensitizers to using hypoxia as a means to selectively target tumors. The more recent description of hypoxia-inducible factors and their role in the hypoxia response network has revealed a host of new drug targets to selectively target tumors.

The Role of Bystander Effects in the Antitumor Activity of the Hypoxia-Activated Prodrug PR-104.

Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment.