Metal-Free CO Prodrugs Activated by Molecular Oxygen Protect against Doxorubicin-Induced Cardiomyopathy in Mice

Figures

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  Figure 1. Organic CO donors capable of CO release under near-physiological conditions without light irradiation. (A) (I) Intermolecular D–A reaction-based CO prodrug; (II) intramolecular D–A reaction-based CO prodrug; (III) elimination-based CO prodrug (LG: leaving group, EWG electro-withdrawing group); (B) oxalic acid-based CO prodrugs using artificial sweeteners as “carriers”; (C) elimination-based CO donors reported by the Larson group; and (D) tautomerization-oxidation-cheletropic reaction-based CO prodrugs described in this work.

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  Figure 2. CO release profiles of QCO-103 (A) and QCO-105 (B) in PBS buffer at different concentrations. The solvent consists of 10% PB and 90% DMSO. The phosphate concentration is indicated in the figure. For (B), phosphate concentration was 10 mM for all groups. (C) UV–vis kinetics profile of QCO-105 with designated phosphate concentrations at pH 5.8 (data interval: 3 min, [QCO] = 50 μM).

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  Scheme 1. Proposed Mechanism of Tautomerization-Induced CO Generation

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  Figure 3. CO release profile in a controlled oxygen environment measured by GC. (A) QCO-103 was incubated in N2 purged DMA, normal DMA, and PB/DMSO (1:9, v/v, pH 10, 10 mM); (B) QCO-105 was incubated in N2 purged PB/DMSO (1:9, v/v, pH 5.8, 10 mM) for 2 h followed by GC analysis of headspace gas. Afterward, 1 mL of air was injected into the headspace vial followed by incubation for 2 h and GC analysis.

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  Figure 4. Cell imaging of CO using the CO probe CODP-202 in macrophages in response to QCO-103 and QCP-103. RAW264.7 cells incubated with (A) 10 μM CODP-202 for 60 min; (B) 10 μM CODP-202 plus 50 μM QCO-103 for 60 min; and (C) 10 μM CODP-202 plus 50 μM QCP-103 for 60 min; scale bar: 25 μm.

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  Figure 5. Transformation of QCO-105 to QCP-105 in RAW264.7 cells (red arrows point to precipitated QCO-105; scale bar: 25 μm).

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  Figure 6. Anti-inflammatory effects of QCO-103 and QCO-105. RAW264.7 cells were pretreated with QCO and QCP compounds at different concentrations for 5 h, followed by the addition of LPS (1 μg/mL) and incubation for 1 h. Culture medium containing 0.5% DMSO was used as the vehicle control (n = 3, **P < 0.01, ***P < 0.001, t-test, TNF is measured by ELISA after 5× dilution of the culture medium).

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  Figure 7. QCO-105 dose-dependently increases the level of HO-1 expression in RAW264.7 macrophages, which was not observed in response to QCO-103 or QCP-105. (A) QCO and QCP at 25 and 50 μM for 6 h; (B) QCO-105 dose-dependently induced HO-1 expression at 6 h (CDDO-Me was used as the positive control).

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  Figure 8. QCO-105 prevents acute doxorubicin (DXR) toxicity to the heart and attenuates the expression of markers of DXR-induced ferroptosis. QCO-105 (50 mg/kg, ip) attenuated DXR (20 mg/kg)-induced cardiac dysfunction after 7 days, evidenced by preserved ejection fraction (A) and fractional shortening (B); (C) representative echocardiography (LVIDd: left ventricular internal dimension in diastole, LVIDs: left ventricular internal dimension in systole); (D) QCO-105 prevented DXR-induced upregulation of the cardiac dysfunction markers β-MyHC and BNP compared to QCP and Solutol controls (3–7 mice per group); and (E) Gpx4 and HO-1 mRNA expression in the ventricle of mice 7 days after a single DXR injection ± QCO-105 (3–5 mice per group, Solutol was used as the vehicle). Expression was determined by RT-qPCR and normalized to that of Hprt1. Mean ± SEM; ns: no significant difference, n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.