Antiviral Chemistry & Chemotherapy 2010 20:201–205 (doi: 10.3851/IMP1480) Short communication Lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice Donald F Smee1*, Brett L Hurst1 and Min-Hui Wong1 1Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA *Corresponding author e-mail: [email protected] Background: Aurintricarboxylic acid (ATA) and ethacrynic 50% cytotoxicity at 84–173 µM, giving low (1.3–4.2) acid (ECA) have been reported to exhibit antiviral activity selectivity index values. Preliminary toxicity tests in against vaccinia virus infections in cell culture by inhib- uninfected mice indicated that ATA and ECA were both iting early and late gene transcription, respectively. The overtly toxic at 100 mg/kg/day. No protection from purpose of this work was to determine if these inhibitors mortality was afforded by treatment of vaccinia virus would effectively treat vaccinia virus infections in mice, infections with ATA or ECA, but 100% survival was which has not previously been studied. achieved in the cidofovir group. ATA- and ECA-treated Methods: ECA was investigated by cell culture plaque reduc- mice died significantly sooner than placebo-treated tion assay for the inhibition of cowpox and vaccinia virus animals, indicating that these compounds exacerbated infections to clarify issues regarding its potency and selec- the infection. tivity. Mice infected intranasally with vaccinia virus were Conclusions: Both ATA and ECA lack antiviral potency treated by intraperitoneal route twice daily for 5 days with and selectivity in cell culture. The compounds were ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) ineffective in treating mice at intraperitoneal doses or once daily for 2 days with cidofovir (100 mg/kg/day). of ≤30 mg/kg/day. These compounds do not appear to Results: ECA caused 50% inhibition of virus plaque have potential for the treatment of poxvirus infections formation at 20–79 µM in four cultured cell lines, with in vivo. Introduction Aurintricarboxylic acid (ATA) and ethacrynic acid 2.5–32 were based upon 50% reductions in virus titre (ECA) have been reported to inhibit vaccinia virus (at 5.4–19.3 µM) rather than on 90% effective con- replication in cell culture [1,2]. ATA inhibits an early centration (EC90 ) values [2]. Both compounds have transcriptional event of vaccinia virus replication by modes of action that differ from nucleotide analogues targeting cellular and viral factors [1]. The compound such as cidofovir that inhibits viral DNA synthesis blocks the phosphorylation of extracellular signal- [3] or the non-nucleoside ST-246 that prevents virus regulated kinase 1/2 (a cellular enzyme) and the phos- assembly [4]. phatase activity of the viral AH1L enzyme. ATA caused The authors of the reports of ATA and ECA inhibition a 90% reduction in virus titre at concentrations of indicate that these molecules might be drug candidates 25–200 µg/ml (60–475 µM), depending upon the cell or provide new directions in pursuing antipoxvirus com- line used, and toxicity was not evident at 500 µg/ml pounds that could be useful as drugs [1,2]. Thus, the (1,185 µM) [1]. ECA was shown to inhibit late viral compounds appear to merit further study, particularly gene expression, with no effect on other virus targets, as no data of their activities in animal models have been such as virus entry into the cell, early gene expression reported. Because of the need for antipoxvirus agents, or viral DNA synthesis [2]. ECA caused a 90% reduc- owing to bioterrorism concerns surrounding the delib- tion in vaccinia virus yield at concentrations of 19–61 erate release of smallpox or monkeypox viruses into µM, depending upon cell type. Cytotoxicity was evi- human populations [5,6], studies to identify new agents dent at 35–290 µM. Selectivity index (SI) values of that have potential for treating humans are warranted. ©2010 International Medical Press 1359-6535 (print) 2040-2066 (online) 201 DF Smee et al. These studies include the determination of compound MK2 and MA-104 cells) foetal bovine serum (FBS). efficacy in animals [7,8]. A549 cells required Ham’s F12K medium with 0.22% From a review of the published literature [1,2], it was sodium bicarbonate and 10% FBS. The medium for all our impression that ATA would be too weak in potency antiviral assays was MEM with bicarbonate, 2% FBS, in vitro to show efficacyin vivo. Although ECA is more and 50 µg/ml of gentamicin. potent than ATA, we were concerned that ECA might be poorly selective (that is, the 50% antiviral and cyto- Plaque reduction assays toxic activities might not be far enough apart). Thus, Sensitivities of cowpox and vaccinia viruses to ECA we performed some cell culture analyses with ECA to were determined in 12-well microplates of cells [9,10]. confirm our hypothesis. We felt that the antiviral activ- The cells were infected with about 75–100 plaque ity of ATA was sufficiently documented not to pursue forming units (PFU) of virus per well, the virus was further in vitro studies with that material. Next, we per- adsorbed for 1 h, then twofold dilutions of antiviral formed animal experiments using intranasally infected compounds were applied for 3 days. Cells were fixed mice treated with ATA and ECA and made comparisons and stained in 5% buffered formalin containing 0.2% with cidofovir treatment. The lack of utility of ATA and crystal violet for 15 min. The aspirated plates were ECA in treating vaccinia virus infections in mice was rinsed with water and plaques were counted. Concen- clearly demonstrated. trations of compounds reducing plaque numbers by 50% (50% effective concentration [EC50] values) were Methods determined by plotting the percentage of plaques rela- tive to untreated cultures versus inhibitor concentra- Antiviral compounds tion on semi-log10 paper. ATA and ECA were purchased from Sigma (St. Louis, MO, USA). Cidofovir was kindly provided by M Hitch- Cytotoxicity assays cock of Gilead Sciences (Foster City, CA, USA). The ECA was overtly toxic to uninfected cells at particular acids were dissolved in 2% sodium bicarbonate solu- concentrations. Percent cytotoxicity was quantified by tions. ECA was further diluted into cell culture medium staining cell monolayers in 12-well plates with crystal for studies conducted in vitro. Prior to discovering a violet after 3 days of ECA treatment. After 15 min, suitable solvent for the compounds (ECA being the first the cells were rinsed of excess dye and the plates were one examined), animal experiments were performed allowed to dry. The dye was eluted from the cells with ECA suspended in saline for treatment of mice. with ethanol and quantified spectrophotometrically ATA was dissolved in 2% sodium bicarbonate buffer at 590 nm [11]. Absorbance values were converted to for mouse experiments. Cidofovir, a positive control, percentages of the untreated control. The 50% cyto- was dissolved in saline. The placebo for animal studies toxic concentration (CC50 value) was determined as was saline for the evaluation of ECA or 2% sodium described above. bicarbonate for the study of ATA. Mouse experiments Viruses and cells Specific pathogen-free BALB/c mice weighing 14–15 g Vaccinia virus IHD and WR strains were purchased were obtained from Charles River Labs (Wilmington, from the American Type Culture Collection (ATCC, MA, USA). Mice were infected intranasally with 50 µl of Manassas, VA, USA). Cowpox virus (Brighton strain) vaccinia (IHD strain) virus (approximately 1×105 PFU/ was obtained from the US Army Medical Research mouse) following anaesthesia with ketamine (100 mg/ Institute of Infectious Diseases (Fort Detrick, Fre- kg, by intraperitoneal [ip] injection). Treatments with derick, MD, USA). The origin of the cowpox virus compounds were given by ip injection starting 24 h was the Centers for Disease Control and Prevention after virus exposure. The treatment schedules and doses (Atlanta, GA, USA). The viruses were initially prop- of antiviral compounds that were used for the experi- agated in African green monkey kidney (Vero) cells ments were based upon our previous work [12]. The (ATCC). Higher virus titres for later use were obtained animals were kept for 21 days to record deaths and to by propagating the viruses in a second line of African monitor body weight. green monkey kidney (MA-104) cells, purchased from BioWhittaker (Walkersville, MD, USA). Other cells Statistical analyses used for in vitro assays included human lung carci- Survival curves of data from mouse studies were noma (A549) and rhesus monkey kidney (LLC-MK2) assessed for all groups by the Mantel–Cox log rank test. cells. The three monkey cell lines were propagated in Because statistical significance among groups was seen minimal essential medium (MEM) containing 0.22% (P<0.001), pairwise comparisons were made using the sodium bicarbonate and 5% (Vero cells) or 10% (LLC- Gehan– Breslow–Wilcoxon test. Evaluations were made 202 ©2010 International Medical Press Antiviral activities of aurintricarboxylic and ethacrynic acids using Prism software (GraphPad Software, San Diego, ECA group. All other groups of ATA- and ECA-treated CA, USA). mice showed increases in body weight. Based on the results of these experiments, it was decided to test each Results compound for antiviral activity at a maximum dose of 30 mg/kg/day. Cell culture experiments Antiviral studies were conducted in mice infected The variability in antiviral potency and SI values with vaccinia virus (IHD strain). This infection evolves reported previously for ECA [2] prompted us to carry slower than infections with the WR strain of vaccinia, out our own investigation into the antiviral activity of and antiviral treatment is more effective [12]. Thus, the compound, using one human and three monkey any positive benefit of treatment with ATA or ECA cell lines.