Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

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  Figure 1:. Comparative analysis of MAP4K4 inhibitors reveals differential effects on H9c2 cell viability in the presence of doxorubicin. (A) Effect of MAP4K4 Inhibitors on the viability of H9c2 Cells. (B) Schematic illustrating viability studies conducted on H9c2 cells treated with doxorubicin and MAP4K4 inhibitors. (C) Viability of H9c2 Cells Treated with Doxorubicin and DMX-5804. (D)Viability of H9c2 cells treated with doxorubicin and GNE-495, (E) Viability of H9c2 cells treated with doxorubicin and MAP4K4-IN3, (F) Viability of H9c2 cells treated with doxorubicin and PF-06260933. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 2:. Effects of DMX-5804 on mitochondrial health of H9c2 cells that have been exposed to doxorubicin. (A) MitoTrackerRed and DAPI staining of healthy, control H9c2 cells. (B) MitoTrackerRed and DAPI staining of H9c2 cells exposed to 10 μM doxorubicin for 24 hours. (C) MitoTrackerRed and DAPI staining of H9c2 cells exposed to 10 μM DMX-5804 for 24 hours. (D) MitoTrackerRed and DAPI staining of H9c2 cells exposed to 10 μM doxorubicin and 10 μM DMX-5804 for 24 hours. (E) Quantification of MitoTrackerRed staining across all four conditions of drug treatment. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001 with one-way ANOVA followed by Dunnett’s multiple comparison’s post-test. (F) TMRM assay of cells exposed to increasing concentrations of doxorubicin alone or increasing concentrations of doxorubicin and 10 μM DMX-5804. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001 with two-way ANOVA followed by Sidak’s multiple comparison’s post-test.

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  Figure 3:. Effects of DMX-5804 on doxorubicin-induced gene expression, drug uptake, and protein signaling in H9c2 cardiomyoblasts. (A) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 4 hours. (B) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 24 hours. RT-qPCR data are shown as mean ± SD from n = 3 independent biological replicates, each measured in duplicate technical replicates. (C) Uptake of 10 μM doxorubicin into H9c2 cells co-treated with 10 μM MAP4K4 inhibitors (DMX-5804, GNE-495, MAP4K4-IN3, and PF-06260933) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA. (D) Uptake of 10 μM doxorubicin into H9c2 cells co treated with increasing concentrations of DMX-5804 (5 μM, 10 μM, and 20 μM) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparison’s post-test. (E) Western blot analysis of H9c2 cells treated with doxorubicin, DMX-5804,…

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  Figure 4:. Effects of MAP4K4 inhibition on doxorubicin uptake, cell viability, and protein signaling in breast cancer cells. (A) Effect of MAP4K4 inhibitors on the cell viability of MDA-MB-231 cells. (B) Uptake of doxorubicin into MDA-MB-231 cells co-treated with MAP4K4 inhibitors after 24 hours. (C) Viability of MDA-MB-231 cells treated with doxorubicin and DMX-5804. (D) Viability of 4T1 cells treated with Doxorubicin and DMX-5804. (E) Western blot analysis of MDA-MB-231 cells treated with doxorubicin, DMX-5804, or both for 24 hours. Data are presented as mean ± stdev, n=4. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

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  Figure 5:. Physicochemical characterization of DMX-5804–loaded liposomal formulations developed for intravenous co-administration with doxorubicin. (A) Size and PDI of liposomal formulations tested. (B) Zeta potential of liposomal formulations. Liposome size, polydispersity index (PDI), and zeta potential are reported as mean ± standard deviation from n = 3 independent liposome preparations.

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  Figure 6:. Effects of NanoDMX on doxorubicin-induced cardiac dysfunction in mice. (A) Ejection fraction of mice treated with doxorubicin, doxorubicin and NanoDMX, doxorubicin and phosphatidylserine free NanoDMX, and relevant vehicle controls. (B) Comparison of ejection fraction of mice at day 0 and day 10, with grey bars representing day 0 and colored bars representing day 10. (C) Comparison of fractional shortening of mice at day 0 and day 10. Data are presented as mean ± standard deviation from n = 5 mice per group and were analyzed using one-way ANOVA followed by Sidak’s multiple-comparisons post hoc test. (D) Ejection fraction over a ten-day period in mice treated with doxorubicin and doxorubicin combined with NanoDMX at increasing doses. (E) Ejection fraction of mice treated with increasing doses of NanoDMX and doxorubicin at day 0 and day 10. (F) Fractional shortening of mice treated with doxorubicin and increasing doses of NanoDMX at day 0 and day 10. Data are presented as mean ± standard deviation from n = 3 mice per group and were analyzed using one-way ANOVA followed by Sidak’s multiple-comparisons post hoc test.

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  Figure 7:. Effects of DMX-5804 delivery on doxorubicin-induced changes in cardiac function and body weight over a 10-day period. (A) Ejection fraction over the 10-day period. (B) Comparison of ejection fraction at baseline (Day 0) and Day 10 across all groups. (C) Comparison of fractional shortening at baseline (Day 0, grey bars) and Day 10 (colored bars) across all groups. (D) Body weight of mice over the ten-day evaluation period following treatment. Data are presented as mean ± standard deviation from n = 5 mice per group and were analyzed using one-way ANOVA followed by Sidak’s multiple-comparisons post hoc test.

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  Figure 8:. Representative H&E-stained sections of the left ventricular free wall from mice receiving doxorubicin with or without DMX-5804 treatment. (A) 20x magnification of left ventricular free wall of healthy mice. (B) 20x magnification of left ventricular free wall of doxorubicin mice. (C) 20x magnification of left ventricular free wall of doxorubicin and orally administered DMX-5804 mice. (D) 20x magnification of left ventricular free wall of doxorubicin and NanoDMX treated mice.