The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-beta-mediated antiviral activity in vitro and in vivo

Abstract

The 2009 outbreak of pandemic H1N1 influenza, increased drug resistance, and the significant delay in obtaining adequate numbers of vaccine doses have heightened awareness of the need to develop new antiviral drugs that can be used prophylactically or therapeutically. Previously, we showed that the experimental anti-tumor drug DMXAA potently induced IFN-β but relatively low TNF-α expression in vitro. This study confirms these findings in vivo and demonstrates further that DMXAA induces potent antiviral activity in vitro and in vivo. In vitro, DMXAA protected RAW 264.7 macrophage-like cells from VSV-induced cytotoxicity and moreover, inhibited replication of influenza, including the Tamiflu®-resistant H1N1 influenza A/Br strain, in MDCK cells. In vivo, DMXAA protected WT C57BL/6J but not IFN-β(-/-) mice from lethality induced by the mouse-adapted H1N1 PR8 influenza strain when administered before or after infection. Protection was accompanied by mitigation of weight loss, increased IFN-β mRNA and protein levels in the lung, and significant inhibition of viral replication in vivo early after DMXAA treatment. Collectively, this study provides data to support the use of DMXAA as a novel antiviral agent.

Figure 1.. Differential induction of IFN-β and TNF-α mRNA expression by DMXAA and LPS in vivo.

C57BL6/J mice were injected i.p. with 0.5 ml saline (n=6), DMXAA (25 mg/kg; ∼500 μg/mouse; n=7), or E. coli LPS (25 μg/mouse; n=7). At 2 h and 5 h (n=3 for each treatment group) after treatment, livers (A) and lungs (B) were extracted, and relative gene expression was analyzed by qPCR and normalized to HPRT. Results represent the mean ± sd.

Figure 2.. DMXAA protects from VSV infection in vitro.

RAW 264.7 cells were treated with DMXAA (100 μg/ml) for 6 h, washed, and infected with VSV with increasing MOIs as indicated. After 24 h, cells were washed with PBS, fixed in 10% buffered formalin, and stained with crystal violet. The results are derived from a single representative experiment (n=3).

Figure 3.. DMXAA inhibits influenza A/Wuhan and Tamiflu^®-resistant influenza A/Br replication in vitro.

Increasing concentration of DMXAA and Tamiflu® was incubated with the indicated viruses (3.0 Log₁₀ TCID₅₀) for 1 h at room temperature and then added to confluent cultures of MDCK cells. Twenty-four hours later, supernatants were collected for measurement of viral titers by the TCID₅₀ assay. Each bar represents the mean of four wells; *P < 0.05.

Figure 4.. IFN-β-dependent, DMXAA-mediated protection of mice against influenza-induced lethality.

(A) DMXAA protects C57BL/6J mice against influenza-induced weight loss. WT C57BL/6J mice were injected i.p. with saline or DMXAA (25 mg/kg; ∼500 μg/mouse). Three hours later, mice were anesthetized with isoflurane and infected i.n. with 50 μl mouse-adapted H1N1 influenza virus (PR8; 200 p.f.u./mouse). Mice received a second i.p. dose of saline or DMXAA (25 mg/kg; ∼500 μg/mouse) on Day 2 after infection. Weight loss was monitored daily for 14 days. Results are compiled from three separate experiments. (B) DMXAA protects C57BL/6J mice against influenza-induced lethality. WT C57BL/6J mice were treated as in A and survival monitored daily for 14 days. Results are compiled from five separate experiments. (C) IFN-β-deficient mice are hypersusceptible to influenza virus-induced lethality. WT C57BL/6J and IFN-β^−/− mice were infected with influenza virus as described in A and survival monitored daily for 14 days. Data are compiled from three separate experiments. (D) DMXAA fails to protect IFN-β^−/− mice from influenza-induced lethality. WT C57BL/6 or IFN-β^−/− mice were treated with saline or DMXAA and infected with influenza virus as described in A. Results are compiled from three separate experiments. The total numbers of mice in each group are shown in parentheses. (E) DMXAA-treated mice have increased IFN-β levels in lung homogenates. C57BL/6J mice were injected i.p. with saline or DMXAA. Three hours later, mice were infected i.n. with 50 μl mouse-adapted H1N1 influenza virus. Mice were killed 3, 24, 48, and 96 h p.i., and lungs were harvested to measure IFN-β protein by ELISA. Results are compiled from two separate experiments (mean±sem). (F) DMXAA provides protection when administered therapeutically. WT C57BL/6J mice were injected i.p. with saline or DMXAA (25 mg/kg; ∼500 μg/mouse) 3 h prior to or 3 h after i.n. infection with mouse-adapted H1N1 influenza virus (PR8; 200 p.f.u./mouse). Mice received a second i.p. dose of saline or DMXAA (25 mg/kg; ∼500 μg/mouse) on Day 2 after infection. Survival was monitored daily for 14 days. Data are compiled from two separate experiments.