Lung cancer, together with head and neck cancer, accounts for more than one-fourth of cancer deaths worldwide. New, non-toxic therapeutic approaches are needed. High-dose IV vitamin C (aka, pharmacological ascorbate; P-AscH⁻) represents a promising adjuvant to radiochemotherapy that exerts its anti-cancer effects via metal-catalyzed oxidation to form H₂O₂. Mn(III)-porphyrins possessing superoxide dismutase (SOD) mimetic activity have been shown to increase the rate of oxidation of AscH⁻, enhancing the anti-tumor effects of AscH⁻ in several cancer types. The current study demonstrates that the Mn(II)-containing pentaazamacrocyclic selective SOD mimetic GC4419 may serve as an AscH⁻/O₂^•− oxidoreductase as evidenced by the increased rate of oxygen consumption, steady-state concentrations of ascorbate radical, and H₂O₂ production in complete cell culture media. GC4419, but not CuZnSOD, was shown to significantly enhance the toxicity of AscH⁻ in H1299, SCC25, SQ20B, and Cal27 cancer cell lines. This enhanced cancer cell killing was dependent upon the catalytic activity of the SOD mimetic and the generation of H₂O₂, as determined using conditional overexpression of catalase in H1299T cells. GC4419 combined with AscH⁻ was also capable of enhancing radiation-induced cancer cell killing. Currently, AscH⁻ and GC4419 are each being tested separately in clinical trials in combination with radiation therapy. Data presented here support the hypothesis that the combination of GC4419 and AscH⁻ may provide an effective means by which to further enhance radiation therapy responses.

The penta-aza macrocyclic manganese compound GC4419 is in phase 3 clinical trials as a modifier of mucositis in H&N cancer treated by radio-chemotherapy based upon its properties as a superoxide dismutase mimetic. In studies to address the potential for tumor radioprotection, a significant anti-tumor effect was identified in tumors generated from the non-small cell lung cancer (NSCLC) cell line H1299, when GC4419 was combined with radiation. This effect was directly related to the size of the radiation dose as demonstrated by greater efficacy in tumor growth delay when biologically equivalent irradiation regimens using a limited number of dose fractions was substantially more effective compared to regimens where the fraction number was higher and dose per fraction decreased. Furthermore, a TCD50 assay using H1299 tumors that tested the combination of GC4419 with radiation revealed a Dose Enhancement Factor of 1.67. Based upon these results the hypothesis that GC4419 was generating cytotoxic levels of hydrogen peroxide during the superoxide dismutation process. Peroxide flux did increase in cells exposed to GC4419 as did the expression of the oxidative stress markers 4-HNE and 3-NT. H1299 cells that overexpressed catalase were then challenged as tumors by the combination of radiation and GC4419 and the tumoricidal effect was nearly eliminated. The enhanced radiation response was not specific to NSCLC as similar findings were observed in human head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma xenografts. RNA sequencing analysis revealed that GC4419, in addition to generating high levels of hydrogen peroxide in irradiated cells, alters inflammatory and differentiation signaling in the tumor following irradiation. Together, these findings provide abundant evidence that the radioprotector GC4419 has dual functionality and will increase tumor response rates when combined with agents that generate high levels of superoxide like stereotactic ablative body radiotherapy (SAbR). Combining SAbR with GC4419 may be an effective strategy to enhance tumor response in general but may also allow for fully potent radiation doses to tumors that might not necessarily be able to tolerate such doses. The potential for protection of organs at risk may also be exploitable.