Renewed interest in using pharmacological ascorbate (AscH−) to treat cancer has prompted interest in leveraging its cytotoxic mechanism of action. A central feature of AscH− action in cancer cells is its ability to act as an electron donor to O2 for generating H2O2. We hypothesized that catalytic manganoporphyrins (MnPs) would increase AscH− oxidation rates, thereby increasing H2O2 fluxes and cytotoxicity. Three different MnPs were tested (MnTBAP, MnT2EPyP, and MnT4MPyP) exhibiting a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP exerted the greatest effect on increasing the rate of AscH− oxidation as determined by the concentration of ascorbate radical [Asc•−] and the rate of oxygen consumption. At concentrations that had minimal effects alone, combining MnPs and AscH− synergized to decrease clonogenic survival in human pancreatic cancer cells. This cytotoxic effect was reversed by catalase, but not superoxide dismutase, consistent with a mechanism mediated by H2O2. MnPs increased steady-state concentrations of Asc•− upon ex vivo addition to whole blood obtained either from mice infused with AscH− or patients treated with pharmacologic AscH−. Lastly, tumor growth in vivo was inhibited more effectively by combining MnT4MPyP with AscH−. We concluded that MnPs increase the rate of oxidation of AscH− to leverage H2O2 flux and ascorbate-induced cytotoxicity.

OBJECT Sentinel headaches (SHs) associated with cerebral aneurysms (CAs) could be due to microbleeds, which are considered a sign that an aneurysm is unstable. Despite the prognostic importance of these microbleeds, they remain difficult to detect using routine imaging studies. The objective of this pilot study is to detect microbleeds associated with SH using a magnetic resonance imaging (MRI) quantitative susceptibility mapping (QSM) sequence and then evaluate the morphological characteristics of unstable aneurysms with microbleeds. METHODS Twenty CAs in 16 consecutive patients with an initial presentation of headache (HA) leading to a diagnosis of CA were analyzed. Headaches in 4 of the patients (two of whom had 2 aneurysms each) met the typical definition of SH, and the other 12 patients (two of whom also had 2 aneurysms each) all had migraine HA. All patients underwent imaging with the MRI-QSM sequence. Two independent MRI experts who were blinded to the patients' clinical history performed 3D graphical analysis to evaluate for potential microbleeds associated with these CAs. Computational flow and morphometric analyses were also performed to estimate wall shear and morphological variables. RESULTS In the 4 patients with SH, MRI-QSM results were positive for 4 aneurysms, and hence these aneurysms were considered positive for non-heme ferric iron (microbleeds). The other 16 aneurysms were negative. Among aneurysm shape indices, the undulation index was significantly higher in the QSM-positive group than in the QSM-negative group. In addition, the spatial averaged wall shear magnitude was lower in the aneurysm wall in direct contact with microbleeds. CONCLUSIONS MRI-QSM allows for objective detection of microbleeds associated with SH and therefore identification of unstable CAs. CAs with slightly greater undulation indices are associated with positive MRI-QSM results and hence with microbleeds. Studies with larger populations are needed to confirm these preliminary findings.

Pharmacological ascorbate (P-AscH - ) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe 3+ ) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe 2+ ). Because paramagnetic Fe 3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH - -induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe 3+ or Fe 2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH - plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH - infusion, post-P-AscH - infusion, and post-radiation (3–4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe 3+ and Fe 2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe 3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe 2+ phantoms. Compared to pre-P-AscH - , GBM T2* and QSM imaging were significantly changed post-P-AscH - infusion consistent with conversion of Fe 3+ to Fe 2+ . No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH - inducing greater changes in tumor labile iron pools compared to the normal tissue. Conclusions T2* and QSM MR-imaging responses are consistent with P-AscH - reducing Fe 3+ to Fe 2+ , selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH - -induced changes in redox-active iron.

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