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Antimicrobial Agents and Chemotherapy, February 2009, p. 572-579, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01257-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Activities of Certain 5-Substituted 4'-Thiopyrimidine Nucleosides against Orthopoxvirus Infections

Earl R. Kern,^1* Mark N. Prichard,¹ Debra C. Quenelle,¹ Kathy A. Keith,¹ Kamal N. Tiwari,² Joseph A. Maddry,² and John A. Secrist III²

Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama,¹ Organic Chemistry Department, Southern Research Institute, Birmingham, Alabama²

Received 19 September 2008/ Returned for modification 22 October 2008/ Accepted 14 November 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4'-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 µM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-iodouracil (4'-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 µM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4'-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log₁₀ units and about 2 log₁₀ units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4'-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.

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The potential use of orthopoxviruses as weapons of bioterrorism and the fact that one member of this group, monkeypox virus, is indigenous in West and Central Africa and has also been introduced through zoonotic spread into the United States have prompted the search for effective and nontoxic antiviral agents for the treatment of orthopoxvirus infections in both animals and humans. Since there is perceived to be little financial reward for the development of a new agent for the treatment of these infections, initial investigations focused on the development of agents that were already approved for use for another indication, such as herpes, AIDS, or hepatitis. One of these agents, cidofovir (CDV), which is approved for use for the treatment of cytomegalovirus retinitis in human immunodeficiency virus-infected patients, is very active in tissue culture cells against all of the orthopoxviruses, including variola virus (2, 7, 8, 9, 13, 15), and has been shown to be highly effective for the treatment of animals experimentally infected with vaccinia, cowpox, ectromelia, and monkeypox viruses (4, 5, 10, 19, 26, 27, 33, 34). Although CDV is approved for use for the emergency treatment of smallpox and complications of vaccination under an experimental Investigational New Drug application, its lack of oral bioavailability somewhat limits its use in a large orthopoxvirus outbreak.

In order to obtain a compound that retains the activity of CDV but that can be administered orally, a series of analogs was synthesized by attaching a long-chain alkoxyalkanol to CDV. One of these compounds, hexadecyloxypropyl CDV (CMX001), was about 100-fold more active than CDV against vaccinia virus and cowpox virus (15, 16). A similar level of enhanced activity of CMX001 against variola and monkeypox viruses has been reported (12, 15). In mice infected with vaccinia, cowpox, or ectromelia virus, CMX001 given orally as either a single dose or multiple doses was at least as effective as CDV, if not more so, in preventing mortality and reducing viral replication in target organs (6, 21, 28). This compound is currently in phase I/II clinical studies for the treatment of orthopoxvirus and herpesvirus infections.

A second compound that is also in phase I/II clinical studies is a low-molecular-weight compound, ST-246, which has potent activity in vitro against all of the orthopoxviruses against which it has been tested (11, 29, 40) and which has been reported to be highly effective when it is given orally in preventing mortality or disease in mice infected with vaccinia, cowpox, or ectromelia virus (29, 40). The compound has a mechanism of action different from that of CDV, and its activity is restricted to poxviruses.

Numerous nucleoside analogs have been reported to be active against vaccinia virus (7). We have evaluated most of the antiviral agents that have been licensed for other uses for their activities against orthopoxviruses (15) and found that CDV, idoxuridine (IDU), and trifluridine have significant activities in vitro without being overtly toxic. The fact that the mechanism of action of IDU against herpes simplex virus involves phosphorylation of the drug by the virus-encoded UL23 thymidine kinase (TK) was of special interest to us, as we have reported that this enzyme is involved in the selective activation of certain inhibitors of orthopoxvirus replication (23-25). In previous studies by Secrist et al. (32) and Rahim and colleagues (31), a series of 2'-deoxy-4'-thiopyrimidine nucleosides were synthesized, and a number of these analogs had significant activity predominantly against the alphaherpesviruses. We subsequently found that one of these analogs, 1-(2'-deoxy-4'-thio-β-D-ribofuranosyl)-5-iodouracil (4'-thioIDU; compound 5 in Fig. 1), also has potent activity in vitro against vaccinia and cowpox viruses. Subsequently, we synthesized and evaluated 16 other analogs for their activities against orthopoxvirus infections.

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FIG. 1. Structures of 5-substituted 4'-thiopyrimidine nucleosides.

The purpose of the studies reported in this communication was to synthesize and evaluate a number of compounds in this series for their activities against vaccinia and cowpox viruses in vitro, investigate their mechanisms of action, determine if they retain their activities against other drug-resistant mutants, and compare their activities against vaccinia and cowpox virus infections in mice to the activity of CDV.

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Synthetic chemistry. 1-(2-Deoxy-4-thio-β-D-ribofuranosyl)-5-fluoro-uracil (Fig. 1, compound 1), 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-thymine (compound 2), 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-bromouracil (compound 3), 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-trifluoromethyluracil (compound 4), and 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-iodouracil (compound 5) were synthesized according to the procedures previously published by Secrist et al. (32) and Rahim et al. (31). 1-(2-Deoxy-4-thio-β-D-ribofuranosyl)-5-phenyluracil (compound 6) was prepared by Suzuki coupling of compound 9 with phenylboronic acid (unpublished results). 1-(4-Thio-β-D-arabinofuranosyl)-5-fluorouracil (compound 7), 1-(4-thio-β-D-arabinofuranosyl)-cytosine (compound 10), 1-(4-thio-β-D-arabinofuranosyl)-5-fluorocytosine (compound 11), 1-(4-thio-β-D-arabinofuranosyl)-5-chlorocytosine (compound 12), and 1-(4-thio-β-D-arabinofuranosyl)-5-methylcytosine (compound 13) were prepared as described by Tiwari et al. (38, 39); and 1-(4-thio-β-D-arabinofuranosyl)-5-iodouracil (compound 8) was synthesized by the same procedure. A prodrug of compound 5 was synthesized by routine acetylation to obtain 1-(2-deoxy-3, 5-di-O-acetyl-4-thio)-5-iodouridine (compound 9). 1-(2-Deoxy-4-thio-β-D-ribofuranosyl)-cytosine (compound 14), 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-bromocytosine (compound 15), 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-chlorocytosine (compound 16), and 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-methylcytosine (compound 17) were prepared by the same methodology described by Secrist et al. (32).

Viruses and cells. Stock pools of vaccinia virus strain Copenhagen and cowpox virus strain Brighton were obtained from John W. Huggins (Department of Viral Therapeutics, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fredrick, MD). Vaccinia virus strain Western Reserve (WR) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The CDV-resistant vaccinia virus strain (CDV^r strain 15A) and the two cowpox virus strains used in the TK assay, crmA lacZ (TK positive [TK⁺]) and TK:GFP lacZ (TK negative [TK^–]), as well as vaccinia virus VVTK::luc, were obtained from Peter C. Turner and Marie N. Becker (University of Florida, Gainesville, FL), and all strains were described previously (1, 3). The mutant ST-246-resistant vaccinia virus (strain VV911) was provided by Robert Jordan (SIGA Technologies, Inc., Corvallis, OR). Working stocks of the vaccinia virus and cowpox virus strains were propagated in Vero cells obtained from ATCC. Human foreskin fibroblast (HFF) cells were prepared as primary cultures from freshly obtained newborn human foreskins. Culture medium for both cell lines was minimal essential medium (MEM) with Earle's salts containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 20 U/ml of penicillin, and 25 µg/ml of gentamicin.

Drug susceptibility assays for vaccinia virus strains and cowpox virus. Plaque reduction and cytopathic effect (CPE) assays were used to determine drug susceptibility, as described previously (14). For the plaque reduction assays, HFF cells were seeded into six-well plates, and the plates were incubated at 37°C with 5% CO₂ and 90% humidity. Two days later, drug at twice the final desired concentration was serially diluted 1:5 in 2x MEM with 10% FBS to provide six concentrations. Aspiration of the culture medium from triplicate wells for each drug concentration was followed by addition of 0.2 ml per well of diluted virus, which gave 20 to 30 plaques per well in MEM containing 10% FBS, or 0.2 ml medium for drug toxicity wells. The plates were incubated for 1 h to allow the virus to adsorb and were shaken every 15 min. An equal amount of 1% agarose was added to each drug dilution, and the mixture was added to each well in 2-ml volumes. Infected monolayers were incubated for 3 days, stained with a solution of neutral red in phosphate-buffered saline (PBS), and incubated for an additional 5 to 6 h. The stain was aspirated, the plaques were counted by using a stereomicroscope at x10 magnification, and the 50% effective concentrations (EC₅₀s) were calculated by standard methods.

Cowpox virus β-galactosidase assay. Dependence on the viral TK was assessed by previously published methods (23, 24). Briefly, monolayers of HFF cells in 96-well plates were incubated at 37°C for 24 h in a humidified incubator. The experimental compounds were then diluted in the plates, and either TK⁺ or TK^– strains of cowpox virus were added at a multiplicity of infection of 0.05 PFU/cell. At 48 h postinfection, the medium was removed and the β-galactosidase substrate chlorophenol red-β-galactopyranoside was added at a final concentration of 50 µg/ml in PBS. The conversion of the colorimetric substrate was determined by measuring the absorbance at 570 nm, and EC₅₀s were calculated by standard methods (21). The ratio of the EC₅₀ for TK^– viruses and the EC₅₀ for TK⁺ viruses was calculated and used as a measure of TK dependence.

DNA synthesis inhibition assays. Monolayers of HFF cells were infected with vaccinia virus at an multiplicity of infection of 3 PFU/cell. After 24 h, the cells were harvested and total DNA was purified with a Wizard DNA purification kit (Promega, Madison, WI). The genome copy number was determined by real-time PCR with primers 5'-CTG CTG TGT GTA TGA AAT GCT TTA AG-3' and 5'-TCT CGG TTT CCT CAC CCA AT-3' and the strain VVTK-specific probe 6-carboxyfluorescein-5'-AGG CTT CCT TTT CTA AAC-3'-6-carboxytetramethylrhodamine (Applied Biosystems, Foster City, CA).

Cytotoxicity assays. (i) Neutral red uptake assay in stationary cells. HFF cells were seeded into 96-well plates at a concentration of 2.5 x 10⁴ cells per well, and the plates were incubated for 24 h. The medium was aspirated; and 125 µl of each drug concentration was added to designated wells in triplicate, while 100 µl of MEM with 2% FBS was added to all other wells. Serial 1:5 dilutions were made with a Beckman BioMek liquid handling system, and the plates were incubated for 7 days in a CO₂ incubator at 37°C. Aspirations of the well contents were followed by the addition of 100 µl/well of a solution of neutral red in PBS and incubation for 1 h. The cells were washed with PBS by using a Nunc plate washer, and 200 µl/well of 50% ethanol containing 1% glacial acetic acid was added. The plates were placed on a rotary shaker for 15 min, and the optical densities at 540 nm were read on a Bio-Tek plate reader. The concentration of drug that reduced cell viability by 50% (CC₅₀) was then calculated.

(ii) Cell proliferation assay. Twenty-four hours prior to assay, HFF cells were seeded into six-well plates at a concentration of 2.5 x 10⁴ cells per well in MEM containing FBS. On the day of the assay, the compounds were diluted serially in medium at increments of 1:5, resulting in concentrations covering a range from 100 to 0.03 µM. The medium from the wells was aspirated, and 2 ml of each drug concentration was added to duplicate wells. The cells were incubated in a CO₂ incubator at 37°C for 3 days. After incubation, the medium-drug solutions were removed, 1 ml of 0.25% trypsin-EDTA was added to each well, and the plates were incubated until the cells started to detach from the plate. Vigorous pipetting was used to obtain a homogeneous cell suspension, 0.2 ml of the mixture was added to 9.8 ml of Isoton III solution, and counts were obtained with a Coulter counter. The counts for each sample were obtained three times, and two replicate wells were used for each sample. The 50% inhibitory concentration (IC₅₀) was calculated by standard methods.

Antiviral activity in mice. Female BALB/c mice (age, 3 weeks) were obtained from Charles River Laboratories, Raleigh, NC. The mice were housed in groups in microisolator cages and were utilized at a quantity of 15 mice per treatment group. They were obtained, housed, utilized, and euthanized according to the regulatory policies of USDA and AAALAC. All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee prior to the initiation of the studies.

Antiviral compounds. CDV (Vistide; Gilead Sciences, Foster City, CA) was diluted in sterile saline to yield the desired doses in a 0.1-ml volume. It was administered as a fine suspension intraperitoneally (i.p.) once daily for 5 days. 4'-ThioIDU was provided as a dry powder, dissolved in a constant amount of dimethyl sulfoxide (DMSO), and then suspended in 0.4% carboxymethyl cellulose (CMC) to yield the desired doses in a 0.1-ml volume for i.p. injection or a 0.2-ml volume for oral gavage. Each dose was administered twice daily for 5 days beginning at various times after viral inoculation. Uninfected mice served as toxicity controls and were treated similarly.

Experimental infections. Vaccinia virus strain WR and cowpox virus infections were initiated by intranasal inoculation of anesthetized (ketamine-xylazine) BALB/c mice with approximately 90% lethal doses, which corresponded to 4 x 10⁴ PFU and 1.6 x 10⁴ PFU for cowpox virus and vaccinia virus, respectively (25, 27). The virus inoculum was instilled into both nostrils with a micropipette at a total volume of 40 µl per animal. For the mortality experiments, the animals were checked at least once daily for 21 days. Pathogenesis studies were performed with both vaccinia virus and cowpox virus in order to determine the effect of 4'-thioIDU on viral replication in the target organs of the mice. Three mice each from the control and the treated groups were euthanized on day 1, 3, 5, 7, or 10 postinoculation for collection of the lung, liver, spleen, and kidney. Samples were pooled by tissue type, homogenized in a 10% (wt/vol) suspension, and frozen until they were assayed for virus. The virus titers were determined by cocultivation of homogenates on Vero cells, and plaques were enumerated after 5 days of incubation.

Statistical evaluations. Mortality rates were analyzed by Fisher's exact test, and the mean day of death was determined by the Mann-Whitney U rank-sum test. A P value of 0.05 was considered significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Compounds active against vaccinia and cowpox viruses. Seventeen 4'-thiopyrimidine nucleosides were synthesized and were initially screened by CPE assays to determine the efficacies of the compounds against both vaccinia and cowpox viruses. Seven of the compounds found to be efficacious by the CPE assays were subject to confirmatory plaque reduction assays, and these results, along with cytotoxicity data, are presented in Table 1. All seven of these compounds had activities against both vaccinia and cowpox viruses that ranged from being 20- to 1,400-fold greater than the activity of CDV. The activity of one compound selected from the series, 4'-thioIDU, was also tested against vaccinia virus WR, and its activities against the Copenhagen strain of vaccinia virus and cowpox virus were compared. Selectivity indices (CC₅₀/EC₅₀) ranged from >200 to 2,000 for 4'-thioIDU; in contrast, the values for CDV were >9 to >32. All of the compounds exhibited minimal toxicity in stationary cells, as measured by the neutral red uptake assay, but cytotoxicity against proliferating cells was considerable (Table 1). The remaining 10 compounds were inactive when they were tested at 100 µM.

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TABLE 1. Cytotoxicities and efficacies of active 4'-thionucleosides against vaccinia and cowpox viruses in HFF cells

Activity against drug-resistant viruses. The antiviral activities of these compounds observed in vitro prompted additional studies of their activities against strains of vaccinia virus that were resistant to CDV or ST-246, and 4'-thioIDU was used as the representative compound from this series. This molecule retained its antiviral activity against a drug-resistant strain of CDV that contained mutations in the E9L DNA polymerase gene (3), as well as a strain resistant to ST-246 that contained mutations in the F13L gene (38) (Table 2). These data indicate that the mechanism of action of this molecule is distinct from the mechanisms of action of CDV and ST-246 and suggest that the molecule might be useful for the treatment of drug-resistant virus infections. The susceptibility of a recombinant virus lacking the J2R gene encoding the viral TK was also evaluated, since 4'-thioIDU is a thymidine analog. This recombinant virus appeared to be comparatively resistant to both 4'-thioIDU and IDU, while it remained fully sensitive to CDV, which does not require phosphorylation by this viral enzyme. These results initially suggested that the mechanism of action of 4'-thioIDU may be similar to that of IDU and that the viral TK may contribute to the phosphorylation of the drug.

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TABLE 2. Activity of 4'-thioIDU against wild-type and resistant mutants of vaccinia virus

Mechanism-of-action studies. Additional studies were conducted to investigate the effects of the drug on viral infection. Previous results from our laboratory showed that the activity of IDU against cowpox virus was dependent on the viral TK, suggesting that 4'-thioIDU might act by a similar mechanism (25). These studies were repeated and IDU exhibited TK dependence, while CDV and ST-246 were equally effective against both strains of virus (Table 3). The antiviral activity of 4'-thioIDU also proved to be largely dependent on the viral TK, which is consistent with our hypothesized mechanism. Subsequent studies examined its effects on viral DNA synthesis and confirmed that it is a potent inhibitor of viral DNA synthesis (Table 3). When they are taken together, the results of these studies suggest that this compound is likely phosphorylated by the viral TK and that at least one of the phosphorylated metabolites affects the synthesis of viral DNA. Nevertheless, the TK-resistant mutants were still partially sensitive to the drug, raising the possibility that other viral or cellular enzymes may also contribute to the phosphorylation of the compound.

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TABLE 3. Role of TK in the activity of 4'-thioIDU against cowpox virus replication

In vivo activity of 4'-thioIDU. To determine if 4'-thioIDU was active in an animal model infection, mice were inoculated intranasally with either vaccinia or cowpox virus. 4'-ThioIDU given i.p. twice daily beginning at 24 h after viral inoculation was highly effective (P < 0.001) at reducing or eliminating mortality when it was used at a dose of 50, 15, or 5 mg/kg of body weight against vaccinia virus or 15, 5, or 1.5 mg/kg against cowpox virus. As a comparison, CDV was used as a positive control and was found to provide complete protection at a dose of 15 mg/kg (Table 4). Since it would be highly desirable for a drug for use for the treatment of poxvirus infections to be active when it was given by the oral route, we next determined the activity of 4'-thioIDU when it was given orally. The drug was administered twice daily beginning at 1, 2, 3, or 4 days after viral inoculation at a concentration of 15, 5, or 1.5 mg/kg and was highly effective (P 0.0001) in preventing mortality against cowpox virus infection when treatment was initiated at as late as 4 days after infection but was ineffective when treatment with 5 mg/kg was begun at 5 days after infection (Table 5). The positive control, CDV, at 15 mg/kg significantly reduced the rate of mortality against cowpox virus infection, even if the initiation of therapy was delayed until 8 days postinfection. In other experiments whose results are not presented here, 4'-thioIDU significantly reduced the rate of mortality when it was given orally at 0.3 or 1.0 mg/kg and when treatment was initiated at 24 h after cowpox virus infection.

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TABLE 4. Effect of i.p. treatment with 4'-thioIDU on mortality of BALB/c mice inoculated intranasally with vaccinia virus strain WR or cowpox virus strain Brighton

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TABLE 5. Effect of oral treatment with 4'-thioIDU on mortality of BALB/c mice inoculated intranasally with cowpox virus strain Brighton

To determine the effect of oral treatment with 4'-thioIDU compared with that of CDV on the replication of vaccinia or cowpox virus in the target organs of mice, animals were inoculated with virus and were treated orally with 15 mg of 4'-thioIDU per kg twice daily beginning at 24 h after infection; CDV was given at 15 mg/kg i.p. once daily. Both of the treatment regimens resulted in significant decreases in the rates of mortality of the control mice (data not shown). In mice infected with vaccinia virus, the virus titers in the liver, spleen, and kidney tissues of both 4'-thioIDU- and CDV-treated mice were reduced to undetectable levels, about a 5-log₁₀-unit reduction, but there was only about a 1-log₁₀-unit decrease in the virus titers in lung tissue (Fig. 2). When mice were infected with cowpox virus, treatment with 4'-thioIDU resulted in a 2-log₁₀-unit reduction in virus titers in the liver, spleen, and kidney tissues (data not shown).

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FIG. 2. Effects of oral treatment with 4'-thioIDU on pathogenesis of vaccinia virus infection in mice.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In addition to the potential threat of the use of an orthopoxvirus, particularly variola virus, as a weapon of bioterror, it is important to emphasize that another member of this group of viruses, monkeypox virus, is endemic in central Africa and is responsible for sporadic outbreaks in the Democratic Republic of the Congo. The ease with which this virus can spread and cause outbreaks in other continents, including North America, was recently demonstrated with the importation of infected animals. It is important, therefore, to regard these agents as potential sources of emerging infections and to continue the goal of developing chemotherapeutic agents effective against these viruses. Although CDV is approved for use for the treatment of smallpox and complications arising from vaccination under an emergency Investigational New Drug application, its lack of activity when it is administered orally and its dose-limiting toxicity would severely limit its use in a large-scale outbreak. To address the need for additional therapies for orthopoxvirus infections, two highly active compounds, CMX001 (a lipid conjugate of CDV) and ST-246 (a small molecule), have advanced through preclinical studies and are being evaluated for their pharmacokinetics and toxicities in clinical studies. Since neither of these compounds has been approved for use for the treatment of orthopoxvirus infections in animals or humans, there continues to be a need for the development of additional therapeutic agents for these infections.

We have investigated a group of 4'-thionucleosides that were reported to have activity against herpes simplex virus, that are related to IDU, and that are likely phosphorylated by virus-encoded TK (30). We were particularly interested in these compounds because of previous observations that IDU and trifluridine were also potent inhibitors of orthopoxvirus replication and that the viral TK phosphorylated these drugs (7, 15, 23-25). While IDU has been tested in mice with vaccinia virus infections (20, 36), the drug was considerably less active than 4'-thioIDU, as we observed in the present study. A total of 17 compounds were synthesized, and 7 of these had activities in vitro against vaccinia and cowpox viruses at concentrations of about 1 µM. Substitution of the pyrimidine base at the 5 position, particularly by halogen, resulted in a high level of activity. One of the first active compounds evaluated in this group was the thymidine analog, 4'-thioIDU; and 4'-thioIDU was used for subsequent experiments, including mechanism-of-action, resistance, and in vivo experiments. While it may not be the most active or the least toxic compound of the group, it has an excellent selectivity index and was not visibly toxic in animals when it was used at 100 times the therapeutic concentration. The in vitro studies indicated that this compound was 30 to 300 times more active against vaccinia and cowpox viruses than CDV, and neither 4'-thioIDU nor CDV was toxic in stationary human cells. It should be pointed out, however, that all of these compounds, including 4'-thioIDU, are considerably more toxic in dividing cells than in nondividing cells.

A major problem with the development of new antiviral agents has been the appearance of resistant mutants, and any new agent should have activity against isolates resistant to existing drugs. Since there has essentially never been a clinical study with an antiviral agent for a disease caused by orthopoxvirus, there is no information regarding the development of resistance to any of these compounds in humans, and no resistant isolates have been recovered in studies of animals treated with any of the agents described above. The development of mutants resistant to CDV and ST-246 in vitro have been reported, although inordinately long passage histories were required to generate resistance to CDV (3, 18, 35, 37, 40). In the present study, we showed that 4'-thioIDU was effective against both CDV- and ST-246-resistant mutants, suggesting that its mechanism of action is distinct from the mechanisms of action of CDV and ST-246. This is important, since the activities of combinations of 4'-thioIDU with either of these drugs might be synergistic and the use of combinations of these drugs may reduce the possibility of the development of resistance (22). We previously reported that CMX001 and ST-246 act synergistically both in vitro and in vivo (30), and the results presented here suggest that 4'-thioIDU might be a good addition to combination therapies with either or both of these drugs. Further studies will be required to document whether synergistic interactions exist between 4'-thioIDU and CMX001 or ST-246, but the potentially different mechanism of action exhibited by 4'-thioIDU makes it a valuable addition to the current set of drugs under development.

While 4'-thioIDU was active against both CDV- and ST-246-resistant mutants of vaccinia virus, it exhibited reduced efficacy against a TK^– strain of vaccinia virus, which suggests that the viral TK is involved in activation of the drug. This is significant, because the selective phosphorylation of this compound by the viral enzyme should result in an increased specificity for infected cells and decreased toxicity. This mechanism of action is distinct from the mechanisms of action of CMX001 and ST-246 and suggests that 4'-thioIDU could be used with either or both of those drugs as part of a combination regimen. Nevertheless, the drug retained significant activity against the TK mutant, which suggests that an additional kinase may be capable of activating the drug or that the compound may be acting by other mechanisms to inhibit the replication of orthopoxviruses and perhaps herpesviruses as well. 4'-ThioIDU clearly inhibited the synthesis of the DNA of the WR strain of vaccinia virus at an EC₅₀ of 0.6 µM (Table 3), which is higher than the efficacy in plaque reduction assays with the same strain (EC₅₀, 0.04 µM; Table 2) but which is comparable to the efficacy in plaque reduction assays with the Copenhagen strain of the virus (EC₅₀, 0.5 µM; Table 1). The differences in EC₅₀s for DNA synthesis and antiviral efficacy that were observed may simply reflect biological variation related to the multiplicity of infection or strain differences, but we cannot exclude the possibility that other targets may be affected by this compound. Additional experiments will be required to determine if 4'-thioIDU inhibits DNA synthesis directly or whether the inhibition of DNA synthesis is a consequence of other defects in the replication cycle.

One of the most rigorous and important determinants of the potential of a new antiviral drug to enter a development path is its ability to reduce virus replication in an experimental animal model of infection. For the initial in vivo evaluation of 4'-thioIDU, mice were inoculated intranasally with either vaccinia or cowpox virus and were treated i.p. with various concentrations of drug by using a standard treatment regimen beginning at 24 h after infection. The drug was highly effective in preventing mortality at concentrations as low as 1.5 mg of drug per kg, which prompted a second series of studies to determine its activity when it was given orally. Importantly, greater than 70% protection was observed even if treatment with concentrations as low as 5 mg per kg was initiated as late as 4 days after infection. These results are equivalent or even superior to those seen with CDV (4, 5, 26, 33, 34), CMX001 (28), and ST-246 (29, 40).

We have previously reported that CDV, CMX001, and ST-246 given parenterally or orally all markedly reduced vaccinia or cowpox virus replication in the liver, spleen, and kidney but resulted in only a small reduction in virus replication in the lung (26-29). This observation is consistent with those of other investigators who have used animal models of vaccinia and cowpox virus infection (4, 34) and also ectromelia virus infection of mice (21). In contrast to these results are the results of treatment with CDV by the intranasal or aerosol route, in which a significant reduction in virus titers in lung tissue has been reported (5, 33). It may be that the high virus titers obtained during vaccinia and cowpox virus infections are more amenable to inhibition by drug delivered directly to the lung than to inhibition of drug given parenterally or orally. Another possibility that may explain the lack of an effect of parenteral or oral delivery in the lung is that the drug does not get to the site of infection. However, in mice inoculated with murine cytomegalovirus, both CDV and CMX001 at concentrations similar to those used in the poxvirus models reduced the virus titers in the lung as well as all of the other target organs by 5 log₁₀ units (17).

4'-ThioIDU is a nucleoside analog that has potent activity against vaccinia and cowpox virus infections in vitro and in vivo. Its activity compares very favorably with those reported for CDV, CMX001, and ST-246; and it is not toxic at therapeutic levels in animals. Its mechanism of action appears to involve its phosphorylation by orthopoxvirus TK, and it inhibits viral DNA synthesis. It is also active against mutants resistant to CDV and ST-246 and may be valuable as part of a combination therapy that includes those drugs. The compound also has activity against the alphaherpesviruses and should continue to be evaluated for a potential role in the treatment of orthopoxvirus and certain herpesvirus infections in humans.

 
We thank Shalisa Sanders and Robin Conley for technical assistance with the in vitro assays and Deborah Collins and Terri Rice for help with the animal studies.

These studies were supported by Public Health Service grant 1-U54-AI-057157 and contracts N01-AI-30049 and N01-AI-15439 to the University of Alabama at Birmingham from the NIAID, NIH.

 
* Corresponding author. Mailing address: Department of Pediatrics, University of Alabama School of Medicine, 128 Children's Harbor Building, 1600 6th Ave. South, Birmingham, AL 35233. Phone: (205) 934-1990. Fax: (205) 975-1992. E-mail: kern{at}uab.edu

Published ahead of print on 24 November 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, February 2009, p. 572-579, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01257-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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