The nucleotide phosphonates cidofovir (CDV) and cyclic cidofovir (cCDV) are potent antiviral compounds when administered parenterally but are not well absorbed orally. These compounds have been reported to have activity against orthopoxvirus replication in vitro and in animal models when administered parenterally or by aerosol. To obtain better oral activity, we synthesized a novel series of analogs of CDV and cCDV by esterification with two long-chain alkoxyalkanols, 3-hexadecyloxy-1-propanol (HDP-CDV; HDP-cCDV) or 3-octadecyloxy-1-ethanol (ODE-CDV; ODE-cCDV). Their activities were evaluated and compared with those of CDV and cCDV in human foreskin fibroblast (HFF) cells infected with vaccinia virus (VV) or cowpox virus (CV) using a plaque reduction assay. The 50% effective concentrations (EC 50 s) against VV in HFF cells for CDV and cCDV were 46.2 and 50.6 M compared with 0.84 and 3.8 M for HDP-CDV and HDP-cCDV, respectively. The EC 50 s for ODE-CDV and ODE-cCDV were 0.20 and 1.1 M, respectively. The HDP analogs were 57-and 13-fold more active than the parent nucleotides, whereas the ODE analogs were 231-and 46-fold more active than the unmodified CDV and cCDV. Similar results were obtained using CV. Cytotoxicity studies indicated that although the analogs were more toxic than the parent nucleotides, the selective index was increased by 4-to 13-fold. These results indicate that the alkoxyalkyl esters of CDV and cCDV have enhanced activity in vitro and need to be evaluated for their oral absorption and efficacy in animal models.Since smallpox was considered to be eradicated in the 1970s, there has been little activity in developing antiviral agents for this infection (10). However, in view of the threat of bioterrorism using variola virus or other orthopoxviruses, such as monkeypox virus, which continues to infect humans in central Africa, there is a renewed need to develop antiviral agents for these viruses (3,11,12,17,18,24). For many years the laboratory of Erik De Clercq and other laboratories have utilized in vitro and animal models with vaccinia virus (VV) to screen potential antiviral compounds for activity against poxviruses and have identified a few active agents. Methisazone, ribavirin, idoxuridine, interferon, interferon inducers, S2442, and cidofovir (CDV) have been identified as potential therapies for these infections (7,8,9,20,21,23). Of particular interest was the finding that CDV and other phosphonate nucleotides were inhibitory to this group of viruses, including VV, cowpox virus (CV), camelpox virus, monkeypox virus, and variola virus (J. W. Huggins, personal communication). The activity of CDV is of particular interest as a potential therapy for smallpox, as it is already approved for the treatment of cytomegalovirus (CMV) infections and has been shown to have activity in animal models using VV and CV (2,22,26,27). We have confirmed the activity of CDV against both VV and CV in tissue culture and animal models in our laboratory (4) and report here the results of some new alkoxyalkyl esters o...

ST-246 was evaluated for activity against cowpox virus (CV), vaccinia virus (VV), and ectromelia virus (ECTV) and had an in vitro 50% effective concentration (EC 50 ) of 0.48 M against CV, 0.05 M against VV, and 0.07 M against ECTV. The selectivity indices were >208 and >2,000 for CV and VV, respectively. The in vitro antiviral activity of ST-246 was significantly greater than that of cidofovir, which had an EC 50 of 41.1 M against CV and 29.2 M against VV, with selectivity indices of >7 and >10, respectively. ST-246 administered once daily by oral gavage to mice infected intranasally with CV beginning 4 h or delayed until 72 h postinoculation was highly effective when given for a 14-day duration using 100, 30, or 10 mg/kg of body weight. When 100 mg/kg of ST-246 was administered to VV-infected mice, a duration of 5 days was sufficient to significantly reduce mortality even when treatment was delayed 24 h postinoculation. Viral replication in liver, spleen, and kidney, but not lung, of CV-or VV-infected mice was reduced by ST-246 compared to levels for vehicle-treated mice. When 100 mg/kg of ST-246 was given once daily to mice infected by the intranasal route with ECTV, treatment for 10 days prevented mortality even when treatment was delayed up to 72 h after viral inoculation. Viral replication in target organs of ECTV-infected mice was also reduced.National preparedness for bioterrorist events includes the development of rapid detection techniques, improved vaccination strategies, and antimicrobial chemotherapeutics with differing modes of action or targets. Preparing for a weaponized variola virus release is one component necessary for national security, and the development of highly effective, nontoxic antiviral agents that have proven efficacy when given postexposure is essential. Although cidofovir (CDV) has been approved under investigational new drug application for treatment of smallpox or complications of vaccination, its use would be limited since it is not orally bioavailable and produces nephrotoxicity.Previous evaluation of ST-246 for activity against orthopoxviruses has shown both in vitro and in vivo efficacies (13). When evaluated in vitro against vaccinia virus (VV), cowpox virus (CV), ectromelia virus (ECTV), monkeypox virus, camelpox virus, and variola virus, ST-246 inhibited virus replication by 50% at a concentration (50% effective concentration [EC 50 ]) of Յ0.07 M. In animal models using lethal infections with ECTV or VV, ST-246 was reported to be nontoxic and effective against mortality when given orally twice daily at 50 mg/kg of body weight for 14 days beginning before or shortly after infection. ST-246 was also evaluated in the nonlethal mouse tail lesion model by use of intravenous VV to mimic the primary viremic phase of viral infection and systemic lesional disease. When ST-246 was administered by oral gavage at 15 or 50 mg/kg twice daily for 5 days, the tail lesions were significantly reduced (13).The current studies expand upon the previous in vitro and in vivo findings and furt...

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The combination of ST-246 and hexadecyloxypropyl-cidofovir or CMX001 was evaluated for synergistic activity in vitro against vaccinia virus and cowpox virus (CV) and in vivo against CV. In cell culture the combination was highly synergistic against both viruses, and the results suggested that combined treatment with these agents might offer superior efficacy in vivo. For animal models, ST-246 was administered orally with or without CMX001 to mice lethally infected with CV. Treatments began 1, 3, or 6 days postinfection using lower dosages than previously used for single-drug treatment. ST-246 was given at 10, 3, or 1 mg/kg of body weight with or without CMX001 at 3, 1, or 0.3 mg/kg to evaluate potential synergistic interactions. Treatment beginning 6 days post-viral inoculation with ST-246 alone only increased the mean day to death at 10 or 3 mg/kg but had no effect on survival. CMX001 alone also had no effect on survival. When the combination of the two drugs was begun 6 days after viral infection using various dosages of the two, a synergistic reduction in mortality was observed. No evidence of increased toxicity was noted with the combination either in vitro or in vivo. These results indicate that combinations of ST-246 and CMX001 are synergistic both in vitro and in vivo and suggest that combination therapy using ST-246 and CMX001 for treatment of orthopoxvirus disease in humans or animals may provide an additional benefit over the use of the two drugs by themselves.

N-Methanocarbathymidine [(N)-MCT] is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. The antiviral activity of this molecule is dependent on the type I thymidine kinase (TK) in herpes simplex virus and also appears to be dependent on the type II TK expressed by cowpox and vaccinia viruses, suggesting that it is a substrate for both of these divergent forms of the enzyme. The drug is also a good inhibitor of viral DNA synthesis in both viruses and is consistent with inhibition of the viral DNA polymerase once it is activated by the viral TK homologs. This mechanism of action explains the rather unusual spectrum of activity, which is limited to orthopoxviruses, alphaherpesviruses, and Epstein-Barr virus, since these viruses express molecules with TK activity that can phosphorylate and thus activate the drug. The compound is also effective in vivo and reduces the mortality of mice infected with orthopoxviruses, as well as those infected with herpes simplex virus type 1 when treatment is initiated 24 h after infection. These results indicate that (N)-MCT is active in vitro and in vivo, and its mechanism of action suggests that the molecule may be an effective therapeutic for orthopoxvirus and herpesvirus infections, thus warranting further development.The most commonly used therapies for herpesvirus infections derive their specificity primarily through selective phosphorylation by viral kinases. Acyclovir (ACV) and penciclovir are phosphorylated by the thymidine kinases (TK) encoded by herpes simplex virus (HSV) and varicella-zoster virus, and the active triphosphate metabolites are potent inhibitors of the viral DNA polymerases (3,5,12,13). These compounds are ineffective against betaherpesviruses since they do not encode TK homologs, yet a closely related nucleoside, ganciclovir (GCV), is specifically phosphorylated by the human cytomegalovirus UL97 kinase (21, 39). Indeed, this strategy could be utilized against any virus provided that (i) a nucleoside could be identified that is a substrate for a viral kinase and (ii) the phosphorylated metabolite is an inhibitor of the viral polymerase. Orthopoxviruses are ideal candidates for such an approach since they encode highly conserved DNA polymerases and they fortuitously express enzymes with TK activity (17). The type II TK homologs in orthopoxviruses differ significantly from the type I homologs encoded by herpesviruses in several respects, including molecular weight, quaternary structure, and a more limited substrate specificity (4). ACV, GCV, and penciclovir are not substrates for this enzyme, and as a consequence, the drugs are inactive against orthopoxviruses. In order to identify new antiviral agents for the treatment of orthopoxvirus infections, we sought to identify nucleoside analogs that were dependent on the cowpox virus (CV) TK for their antiviral activity and, by inference, should be selective inhibitors of this virus and other viruses that express enzymes with TK activity. Herein, we de...