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

Abstract

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-N-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). SN30000, unlike doxorubicin, cisplatin, gemcitabine or paclitaxel, was more active against cells in spheroids than monolayers by clonogenic assay. Combinations of SN30000 and chemotherapy drugs in HCT116/GFP and SiHa spheroids demonstrated hypoxia-and schedule-dependent potentiation of gemcitabine or doxorubicin in growth inhibition and clonogenic assays. Co-administration with SN30000 suppressed clearance of gemcitabine in NIH-III mice, likely due to SN30000-induced hypothermia which also modulated extravascular transport of gemcitabine in tumor tissue as assessed from its diffusion through HCT116 multicellular layer cultures. Despite these systemic effects, the same schedules that gave therapeutic synergy in spheroids (SN30000 3 h before or during gemcitabine, but not gemcitabine 3 h before SN30000) enhanced growth delay of HCT116 xenografts without increasing host toxicity. Identification of hypoxic and S-phase cells by immunohistochemistry and flow cytometry established that hypoxic cells initially spared by gemcitabine subsequently reoxygenate and re-enter the cell cycle, and that this repopulation is prevented by SN30000 only when administered with or before gemcitabine. This illustrates the value of spheroids in modeling tumor microenvironment-dependent drug interactions, and the potential of HAPs for overcoming hypoxia-mediated drug resistance.

Patient-Derived Xenograft and Organoid Models for Precision Medicine Targeting of the Tumour Microenvironment in Head and Neck Cancer

“…Patient tumour specimens can be contaminated by non-tumour cells which, when the specimens are grown as organoids, can outcompete the tumour cells and limit the effectiveness of the organoids at modelling human cancer [ 151 ]. Organoids have been reported to recapitulate hypoxic gradients [ 152 ]; however, their use for evaluating hypoxia-targeting drugs is challenging, since addition of the drug to the culture media causes physical disturbance that alters oxygen gradients in the media and will change the hypoxia status of the organoids [ 153 ]. Finally, a hallmark of cancer cells is anchorage-independent growth, yet, unlike spheroids or in vivo tumour models, tumour organoids are anchored to a basement membrane matrix.…”

Medicinal Chemistry of Drugs with N-Oxide Functionalities

“…Studies highlight its heightened activity on tumor spheroids, leading to significant tumor growth delay when combined with radiation . In addition, SN30000, when used with gemcitabine, effectively inhibits the proliferation of reoxygenated tumor cells . The binding of EF5 may serve as a promising biomarker for hypoxia stratification and the assessment of SN30000 treatment response .…”

“… 135 In addition, SN30000, when used with gemcitabine, effectively inhibits the proliferation of reoxygenated tumor cells. 136 The binding of EF5 may serve as a promising biomarker for hypoxia stratification and the assessment of SN30000 treatment response. 137 It was found that the substitution pattern of the heterocyclic scaffold influences the back-oxidation of the intermediately formed radical anion (e.g., TPZ •– ) and thus the selective hypoxic release of hydroxyl radicals.…”

Targeting Hypoxia: Hypoxia-Activated Prodrugs in Cancer Therapy

“…( 188 ) proved that, compared with monolayer tumor cells, SN30000 has higher activity on tumor spheroids, and when combined with radiation, it can cause significant tumor spheroid growth delay. Moreover, when used together with or before gemcitabine, SN30000 can effectively inhibit the proliferation of reoxygenated tumor cells ( 189 ). EF5 binding may be a promising biomarker for hypoxia stratification and SN30000 treatment response assessment ( 190 , 191 ).…”