Craig W. Day

Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza

Blunting increased vascular permeability caused by an infection-induced cytokine storm with a Slit ligand increased survival in rodent models of sepsis and viral infection. Batten Down the Vascular Hatches Against the Storm An organism under stress from a massive infection or burn reacts strongly to protect itself. Body-wide inflammation is triggered, but this response can have negative effects of its own. These can include a fast heart rate, abnormally high temperature, and a marked discharge of cytokines from the immune system, called a cytokine storm. Death often occurs in these patients, a result of the failure of multiple organs. In mice, London et al. now inhibit one of the consequences of a cytokine storm—leakage from the vasculature into the intercellular space—and can thereby prevent the lethal effects of bacterial and viral infection. Cytokines released during a cytokine storm, including tumor necrosis factor and interleukin-1β, act on the cells of the vascular lining, weakening their junctions and allowing cells and fluid to leak in. Because the resulting edema is one cause of organ failure, London et al. have targeted this process. They show that an abbreviated version of a soluble ligand, called Slit, when added to cultured vascular endothelial cells, can strengthen the cell-cell contacts. Acting by increasing the amount of a cell adhesion protein, vascular endothelial cadherin, on the cell surface, Slit reduced the permeability of the endothelial cell layer. This was also true in whole animals; the authors injected mice with an immunogenic bacterial protein to simulate infection and then measured vascular leakage. They then tested whether Slit could help mice survive a severe infection. Whether infected with gut bacteria or H5N1 flu, treatment of the mice with the abbreviated Slit molecule improved their odds of surviving. Further, the ability of Slit to reduce vascular permeability in the face of a severe infection depended on another signaling molecule, the Robo4 receptor. Sepsis and other illnesses in which a cytokine storm is triggered are difficult to treat effectively. The standard of care is rapid antibiotic administration and supportive treatment of patients, but this is too often ineffective. The approach described here by London et al. may yield another tool to fight the cytokine storm, a way to strengthen the ability of the body to withstand its own assault. The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1β and tumor necrosis factor–α, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host’s immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host’s response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host’s innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Efficacy of Orally Administered T-705 on Lethal Avian Influenza A (H5N1) Virus Infections in Mice

ABSTRACT T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 μM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO2), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO2. Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.

Effect of Interferon-Alpha and Interferon-Inducers on West Nile Virus in Mouse and Hamster Animal Models

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies. Studies were conducted in cell culture and in rodent animal models to determine the efficacy of interferon-alpha (IFN-α), interferon (IFN) inducers and ribavirin, alone or in combination with IFN, in treating WNV. Intraperitoneal injection of IFN-α B/D (qd for 7 days), polyIpolyC(12)U [Ampligen (every other day for 7 days)] and topically applied imiquimod (qd for 7 days), administered from 1 day before viral challenge, were effective in protecting, respectively, 100%, 100% and 70% of BALB/c mice from mortality induced by subcutaneous injection of WNV. When IFN-α B/D or Ampligen treatments were delayed to 4–6 h before viral challenge in mice, efficacy was greatly diminished. Infected Syrian golden hamsters treated with interferon alphacon-1 (Infergen) and Ampligen 4–6 h before viral challenge gained more weight and had a greater survival than saline-treated animals. A combination study of subcutaneously administered Infergen (5 to 0.05 μg/kg/day) and ribavirin (75 to 7.5 mg/kg/day) in >7 week old hamsters demonstrated that Infergen was slightly efficacious in reducing mortality and disease signs; however, it was not synergistic in its antiviral effects when combined with ribavirin. Ribavirin treatment alone increased mortality of infected hamsters. The reduced mortality correlated with reduced plasma viraemia. Since WNV-infected patients have already been treated with IFN and ribavirin and future clinical trials have been suggested, this first report of IFN alone or in combination with ribavirin in WNV-infected animal models might provide useful information for subsequent treatment of patients.

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

Abstract Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572–3577). Ribavirin given at 75mg/kg 4h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1α, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Evaluation of Immunomodulators, Interferons and Known in Vitro SARS-CoV Inhibitors for Inhibition of SARS-Cov Replication in BALB/c Mice

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid interferon, interferon alpha (IFN-α) B/D, and a mismatched double-stranded (ds) RNA interferon (IFN) inducer, Ampligen® (poly I:poly C124), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-α B/D once daily for 3 days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log10 at 10,000 and 32,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen® used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, β-D-N4-hydroxycytidine, calpain inhibitor VI, 3-deazaneplanocin A and Alferon® (human leukocyte IFN-α-n3) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-α can inhibit SARS-CoV replication in the lungs of mice.

A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Abstract Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Treatment of Venezuelan equine encephalitis virus infection with (−)-carbodine

Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(-) enantiomer of carbodine [(-)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 microg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (-)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kgd) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (-)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (-)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (-)-carbodine is effective when initiated after the establishment of brain infection.

D282, a Non-Nucleoside Inhibitor of Influenza Virus Infection that Interferes with de novo Pyrimidine Biosynthesis

Background: The discovery of novel influenza virus inhibitors remains an important priority in light of the emergence of drug-resistant viruses. Toward this end, a library of over 6,000 compounds was tested for antiviral activity. Methods: Strains of influenza virus were evaluated by cytopathic effect (CPE) inhibition and virus yield reduction assays. Intracellular nucleoside triphosphate pools were analysed by strong anion exchange HPLC. Dihydroorotate dehydrogenase inhibition assays were conducted. Influenza virus-infected mice were treated for 5 days with D282. Results: A non-nucleoside, 4-[(4-butylphenyl)amino]-2-methylene-4-oxo-butanoic acid (D282), was discovered that inhibited influenza A and B virus CPE by 50% at 6–31 μM (giving selectivity indices of >13 to >67, based on cytotoxicity of >400 μM in stationary cell cultures). Ribavirin (positive control) was active at 14–44 μM (yielding selectivity indices of >9 to >29, with >400 μM toxicity). D282 and ribavirin inhibited virus yield by 90% at 9.5 ±3.3 and 10.8 ±3.2 μM, respectively. The antiviral activity of D282 in vitro was reversed by addition of uridine, cytidine and orotic acid. D282 exhibited an uncompetitive inhibition of mouse liver dihydroorotate dehydrogenase (inhibitor constant [Ki] of 2.3 ±0.9 μM, Michaelis constant [Km] of 150 ±16 μM). Because cellular pyrimidine biosynthesis was inhibited, D282-treated cells had decreased uridine triphosphate and cytidine triphosphate levels. D282 (≤100 mg/kg/day) failed to prevent death of mice infected with influenza. Conclusions: D282 was active against influenza A and B viruses by inhibiting de novo pyrimidine biosynthesis. Although effective in vitro, the compound, like others in its class, was devoid of antiviral activity in infected mice.

Novel 3-Sulphonamido-Quinazolin-4(3H)-One Derivatives: Microwave-Assisted Synthesis and Evaluation of Antiviral Activities against Respiratory and Biodefense Viruses

We designed and synthesized novel 2,3-disubstituted quinazolin-4(3H)-ones by microwave technique and characterized them by spectral analysis. Synthesized compounds were screened for cytotoxicity and for antiviral activity against influenza A (H1N1, H3N2 and H5N1), severe acute respiratory syndrome corona, dengue, yellow fever, Venezuelan equine encephalitis (VEE), Rift Valley fever, and Tacaribe viruses in cell culture. A neutral red uptake assay was used to determine 50% virus-inhibitory concentrations (EC50) of test compounds and their 50% cytotoxicity concentration (CC50) in uninfected Madin-Darby canine kidney, Vero, and Vero 76 cells; selectivity indices (ratio of CC50 to EC50) were derived from the data. The compound 4-(6,8-dibromo-4-oxo-2-phenyl quinazolin-3(4H)-yl)-N-(4,5-dimethyloxazol-2yl) benzenesulphonamide 15 inhibited the replication of avian influenza (H5N1) virus (EC50=8.4 µg/ml, CC50>100 µg/ml, SI>11.9) as did 4-(6-bromo-4oxo-2phenylquinazolin-3(4H)-yl) benzene]sulphonamide 5 (EC50=3 µg/ml, CC50=32 µg/ml, SI=11). Compound 5 was also moderately active against VEE and Tacaribe viruses. The methodology described in this report is applicable for rapid synthesis of many compounds with potential antiviral properties.

Antivirally active ribavirin analogues--4,5-disubstituted 1,2,3-triazole nucleosides: biological evaluation against certain respiratory viruses and computational modelling.

Background: Ribavirin is a broad-spectrum antiviral agent that derives some of its activity from inhibition of cellular inosine monophosphate dehydrogenase (IMPDH), resulting in lower guanosine triphosphate (GTP) levels. Here we report the biological activities of three ribavirin analogues. Methods: Antiviral activities of test compounds were performed by in vitro cytopathic effect inhibition assays against influenza A (H1N1, H3N2 and H5N1), influenza B, measles, parainfluenza type 3 (PIV-3) and respiratory syncytial viruses. Compounds were modelled into the ribavirin 5‘-monophosphate binding site of the crystallographic structure of the human type II IMPDH (hIMPDH2) ternary complex. Effects of compounds on intracellular GTP levels were performed by strong anion exchange HPLC analysis. Results: Of the three compounds evaluated, the 5-ethynyl nucleoside (ETCAR) exhibited virus-inhibitory activities (at 1.2–20 μM, depending upon the virus) against most of the viruses, except for weak activity against PIV-3 (62 μM). Antiviral activity of ETCAR was similar to ribavirin; however, cytotoxicity of ETCAR was greater than ribavirin. Replacing the 5-ethynyl group with a 5-propynyl or bromo substituent (BrCAR) considerably reduced antiviral activity. Computational studies of ternary complexes of hIMPDH2 enzyme with 5‘-monophosphates of the compounds helped rationalize the observed differences in biological activity. All compounds suppressed GTP levels in cells; additionally, BrCAR suppressed adenosine triphosphate and elevated uridine triphosphate levels. Conclusions: Three compounds related to ribavirin inhibited IMPDH and had weak to moderate antiviral activity. Cytotoxicity adversely affected the antiviral selectivity of ETCAR. As with ribavirin, reduction in intracellular GTP may play a role in virus inhibition.