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 gH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 F 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-01pancreatic tumors and docetaxel with 22RV1prostate 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.Hypoxia is a uniquely attractive target in oncology for two reasons. The first is that hypoxic cells are obstacles to curative cancer therapy with all major treatment modalities. Hypoxia can compromise outcomes of surgery by increasing tumor metastasis (1 -3). It is also a major cause of radioresistance because oxygen is a radiosensitizer, and multiple clinical studies have documented the importance of hypoxia determining local tumor control in radiotherapy (4 -6). Hypoxia also contributes to chemoresistance through multiple mechanisms (7), including limitations on delivery of blood-borne drugs to hypoxic regions of tumors (8,9). The second reason for targeting hypoxia is that it is a common feature of a wide variety of human tumors and is typically more severe in tumors than in normal tissues, thus providing a basis for tumor selectivity (10,11).Several strategies for exploiting tumor hypoxia are now in preclinical or clinical development (7), with the main focus on prodrugs that are activated by metabolic reduction under hypoxic conditions to form cytotoxins. Early efforts focused on quinone bioreductive drugs, such as porfiromycin (12), and 2-nitroimidazole -linked alkylating a...
Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxiaactivated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents. Cancer Res; 75(19); 4211-23. Ó2015 AACR.
Intratumoral hypoxia occurs in many solid tumors, where it is associated with the development of metastatic character. However, the connections between these phenomena are not fully understood. In this study, we define an integrative role for the E3 ubiquitin ligase subunit WSB1. In primary osteosarcomas, increased levels of WSB1 correlated with pulmonary metastatic potential. RNAi-mediated attenuation of WSB1 or disruption of its E3 ligase activity potently suppressed tumor metastasis. Quantitative proteomic and functional analyses revealed that WSB1 ubiquitylates the Rho-binding protein RhoGDI2 and promotes its proteasomal degradation, thereby activating Rac1 to stimulate tumor cell motility and invasion. Our findings show how WSB1 regulates key steps of the metastatic cascade in hypoxia-driven osteosarcoma, and they highlight a candidate therapeutic target to potentially improve the survival of patients with metastatic disease. Cancer Res; 75(22); 4839-51. Ó2015 AACR.
Das hypoxämische Ziel im Blick: Die Kombination einer Nitro‐Wirkstoffvorstufe mit einem wasserlöslichen Phosphat wandelt Duocarmycin‐Analoga von hoch toxischen DNA‐Alkylierungsmitteln in hoch selektive Tumorwirkstoffe um. Diese Wirkstoffvorstufen (siehe Schema) sind in vivo außergewöhnlich wirksam gegen hypoxämische Tumorzellen – die Zellen, die allgemein als am schlechtesten durch herkömmliche Therapiemethoden erreichbar gelten.
ABSTRACTinjected with primary leukemia cells.
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. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.
BackgroundThe phosphate ester PR-104 is rapidly converted in vivo to the alcohol PR-104A, a nitrogen mustard prodrug that is metabolised to hydroxylamine (PR-104H) and amine (PR-104M) DNA crosslinking agents by one-electron reductases in hypoxic cells and by aldo-keto reductase 1C3 independently of oxygen. In a previous phase I study using a q 3 week schedule of PR-104, the maximum tolerated dose (MTD) was 1100 mg/m2 and fatigue, neutropenic fever and infection were dose-limiting. The primary objective of the current study was to determine the dose-limiting toxicity (DLT) and MTD of weekly PR-104.MethodsPatients with advanced solid tumours received PR-104 as a 1-hour intravenous infusion on days 1, 8 and 15 every 28 days with assessment of pharmacokinetics on cycle 1 day 1. Twenty-six patients (pts) were enrolled (16 male/10 female; median age 58 yrs, range 30 to 70 yrs) who had received a median of two prior chemotherapy regimens (range, 0 to 3) for melanoma (8 pts), colorectal or anal cancer (3 pts), NSCLC (3 pts), sarcoma (3 pts), glioblastoma (2 pts), salivary gland tumours (2 pts) or other solid tumours (5 pts). PR-104 was administered at 135 mg/m2 (3 pts), 270 mg/m2 (6 pts), 540 mg/m2 (6 pts), 675 mg/m2 (7 pts) and 900 mg/m2 (4 pts) for a median of two treatment cycles (range, 1 to 7 cycles) and five infusions (range, 1 to 18) per patient.ResultsDose-limiting toxicities (DLTs) during cycle one included grade four thrombocytopenia at 540 mg/m2 (1 of 6 pts) and grade four thrombocytopenia and neutropenia at 900 mg/m2 (2 of 4 pts). At an intermediate dose of 675 mg/m2, there were no DLTs among a total of seven patients given 12 treatment cycles but all experienced moderate to severe (grade 2 to 4) haematological toxicity. Thrombocytopenia was delayed in its onset and nadir, and its recovery was protracted and incomplete in many patients. There were no complete or partial tumour responses. PR-104-induced thrombocytopenia and neutropenia correlated with plasma AUC of PR-104, PR-104A and an oxidative semi-mustard metabolite (PR-104S1), but no more strongly than with PR-104 dose-level. There was no significant correlation between plasma AUC for the reduced metabolites and myelotoxicity.ConclusionsThrombocytopenia, and to a lesser extent neutropenia, was the DLT of weekly PR-104. The MTD was 675 mg/m2/week. PR-104 given weekly may be a suitable protocol for further clinical evaluation as a short course of treatment with fractionated radiotherapy or haematopoietic stem cell support, as its duration of dosing is restricted by delayed-onset and protracted thrombocytopenia.
Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective cytotoxin that has shown significant activity in advanced clinical trials in combination with radiotherapy and cisplatin. The current study aimed to advance our understanding of tirapazamine-induced lesions and the pathways involved in their repair. We show that homologous recombination plays a critical role in repair of tirapazamine-induced damage because cells defective in homologous recombination proteins XRCC2, XRCC3, Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with the involvement of homologous recombination repair, we observed extensive sister chromatid exchanges after treatment with tirapazamine. We also show that the nonhomologous end-joining pathway, which predominantly deals with frank double-strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In addition, we show that tirapazamine preferentially kills mutants both with defects in XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable DNA-topoisomerase I cleavable complexes. We further show that ;H2AX, an indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle. These observations lead us to an overall model of tirapazamine damage in which DNA single-strand breaks, base damage, and DNA-protein cross-links (including topoisomerase I and II cleavable complexes) produce stalling and collapse of replication forks, the resolution of which results in DSB intermediates, requiring homologous recombination and XPF/ERCC1 for their repair. [Cancer Res 2008;68(1):257-65]
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