Review Progress in the discovery of compounds inhibiting orthopoxviruses in animal models

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1 Antiviral Chemistry & Chemotherapy 19: Review Progress in the discovery of compounds inhibiting orthopoxviruses in animal models Donald F Smee Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA Corresponding author: dsmee@cc.usu.edu Surrogate animal models must be used for testing antiviral agents against variola (smallpox) virus infections. Once developed, these compounds can be stockpiled for use in the event of a bioterrorist incident involving either variola or monkeypox virus, or used to treat an occasional serious orthopoxvirus infection, such as disseminated vaccinia complication following exposure to the live virus vaccine. Recently, considerable progress has been made in the discovery of novel antiviral agents found active against orthopoxviruses in vivo. This includes the development of new animal models or refinement of existing ones for compound efficacy testing. Current mouse models employ ectromelia, cowpox and vaccinia (WR and IHD strains) viruses with respiratory (lung) or tail lesion infections commonly studied. Rabbitpox and vaccinia (WR strain) viruses are available for rabbit infections. Monkeypox and variola viruses are used for infecting monkeys. This review describes these and other animal models, and covers compounds found active in vivo from 2003 to date. Cidofovir, known to be active against orthopox virus infections prior to 2003, has been studied extensively over recent years. New compounds showing promise are orally active inhibitors of orthopoxvirus infections that include ether lipid prodrugs of cidofovir and (S)-HPMPA, ST 246, N methanocarbathymidine (N MCT) and SRI (a 4 thio derivative of iododeoxyuridine). Another compound with high activity but requiring parenteral administration is HPMPO-DAPy. Further development of these compounds is warranted. Introduction Although variola virus, the etiological agent in smallpox infection, was declared eradicated from the earth in 1980, the reality of using variola virus and the related monkeypox virus as bioweapons is apparent [1 4]. Hence, in the 1990s the US government began pursuing the goal of identifying at least two orthopoxvirus inhibitors, ideally with different modes of antiviral action, that could be stockpiled and used as approved drugs for treatment of infections causes by these or other orthopoxviruses. A vigorous research effort in the USA and abroad commenced, and involved pioneering work at the US Army Medical Research Institute of Infectious Diseases (USAM- RIID) at Fort Detrick, Maryland. Active and potent compounds against variola virus were identified by cell culture screening [5], and new animal models were developed using cowpox and monkeypox viruses in mice and monkeys, respectively [6 8]. An extensive review of the research performed on anti-orthopoxviruses found to be active in animal models from 1950 to 2002 was published in 2003 [9]. Since 2002, considerable progress has been made in anti-orthopoxvirus research. Many new animal models have been developed, genetically modified orthopoxviruses applicable to the work have been prepared, and important new compounds have been synthesized and found active in animal models. Because of these developments, it was deemed appropriate to write an update in the form of this review, covering the period from 2003 to the present time. An attempt was made to include all published sources of compounds that have shown efficacy in animal models of orthopoxvirus infection. Pertinent published abstracts were also included, when a full research paper had not yet been published. A brief summary of the previous work [9] has also been given to bring the reader to the point where this review begins. Orthopoxvirus animal models used for chemotherapy studies between Mice have primarily been used for chemotherapy studies of orthopoxvirus infections prior to International Medical Press

2 DF Smee Table 1. Animal models used for evaluating antiviral agents against poxviruses from Animal species Virus Inoculation route Primary infection* References Mouse Rabbitpox, vaccinia, variola ic Encephalitis [24 27] Mouse Cowpox, cowpox CDV-R, vaccinia Aerosol or in Pneumonitis [6,7,25,28,29] Mouse (SCID) Cowpox, cowpox CDV-R, vaccinia in or iv Pneumonitis [6,29 31] Mouse (immunosuppressed) Vaccinia iv Systemic [32] Mouse Vaccinia Tail scarif or iv Tail lesions [33,34] Rabbit Rabbitpox in Pneumonitis [33] Rabbit Vaccinia Eye scarif Keratitis [35,36] Rabbit Vaccinia Skin scarif Skin lesions [37,38] Monkey Vaccinia Eye scarif Keratitis [39] Monkey Vaccinia Skin scarif Skin lesions [40] Monkey Monkeypox Aerosol Pneumonitis, skin lesions [8,41] *Some skin infections and pneumonitis progressed to being systemic. For a complete reference list for these models see Smee and Sidwell [9]; the earliest dates were cited for each model. Tail lesions also develop with intravenous (iv) inoculations. CDV-R, cidofovir-resistant; ic, intracerebral; in, intranasal; scarif, scarification; SCID, severe combined immunodeficient. (Table 1). Mice infected intracerebrally with rabbitpox, vaccinia and variola viruses developed encephalitis. However, these models have not been used since 1979 for antiviral studies [9]. More popular models in mice used during the time period were intranasal or aerosol challenges with cowpox and vaccinia viruses causing pneumonitis, scarification or intravenous infections producing tail lesions, and intraperitoneal injections (such as those in severe combined immunodeficient [SCID] mice) that lead to systemic disease and death. A cidofovir (CDV)-resistant cowpox virus was developed and studied in mouse pneumonitis and SCID mouse models. Ectromelia (mousepox) virus was known to cause severe fatal infections in mice [10,11]. Antiviral studies using this infection model began to appear in the published literature after Antiviral treatments of rabbitpox and vaccinia virus infections in rabbits have been conducted (Table 1). Intracerebral injections caused encephalitis, eye scarification caused keratitis, skin scarification caused lesion formation (pocks) on the skin, and intranasal instillation caused pneumonitis (with rabbitpox virus). Antiviral studies in monkeys have been few (Table 1) because of high costs and limited numbers of animals. Monkeys scarified in the eyes and skin with vaccinia virus developed keratitis and skin lesions, respectively. Aerosol infection of monkeys with monkeypox virus has served as a surrogate model for studying the antiviral treatment of variola (smallpox) virus infection. Prior to 2003 the search was on to identify a potential model for variola virus in monkeys, work that reached fruition after that time. Other types of orthopoxvirus infections are known to occur in their natural host, such as those caused by buffalopox, camelpox, raccoonpox, taterapox, volepox and African horsepox virus [9]. There have been no reported small animal models for these infections. Newly reported or refined orthopoxvirus animal models since 2002 With greater emphasis on the discovery and development of antiviral agents against orthopoxvirus infections has come a plethora of new animal models, or refinement of existing ones. As might have been expected, the variety of new mouse models has surpassed those of other animal species (Table 2). Antiviral studies were originally conducted with ectromelia virus in mice, primarily using respiratory infection routes, although footpad injection can also be used as a model. A highly lethal ectromelia virus was developed by inserting the interleukin-4 (IL-4) gene into it, resulting in immune dysfunction in the host. New parameters were investigated in mouse models, such as determining cytokine and chemokine profiles in animals infected either intranasally or intraperitoneally with cowpox and vaccinia viruses. Whole body imaging had been reported following infection with cowpox virus, producing green fluorescent protein. Lethal and non-lethal strains of CDV-resistant (CDV-R) vaccinia virus were developed and used for chemotherapy studies. The vaccinia virus IHD strain seems to have gained favour over using vaccinia WR strain for many chemotherapy studies because its virulence is less, making infections with the IHD strain treatable with lower doses of active compounds. Infection with a lethal dose of cowpox virus in a small volume (5 µl) led principally to a fatal upper respiratory infection, with minimal lung infection or dissemination to other organs. This is in contrast to higher volume (50 µl) inoculations of the same infectious dose that caused severe pneumonitis and death. Cutaneous infection models have been developed using immunocompetent or immunosuppressed hairless mice or athymic nude mice. Novel small animal species have been used or are being studied as models for the treatment of monkeypox International Medical Press

3 Orthopoxvirus inhibitors in animals Table 2. Newly reported or further refined animal models used for evaluating antiviral agents against poxviruses since 2002 Primary infection* Animal species Virus Inoculation route (refinement of model) References Mouse Ectromelia Aerosol, in Pneumonitis [42,43] Mouse Ectromelia Footpad inj Systemic [44] Mouse Ectromelia IL-4 Footpad inj Systemic [45] Mouse Cowpox, vaccinia in Pneumonitis [46] (cytokine and chemokine profiles) Mouse Cowpox, vaccinia ip Systemic [46] (cytokine and chemokine profiles) Mouse Cowpox-GFP ip Systemic [47] (whole body imaging) Mouse Vaccinia CDV-R in Pneumonitis [48,49] non-lethal strains Mouse Vaccinia CDV-R in Pneumonitis [50] lethal strain Mouse Vaccinia IHD strain in Pneumonitis [51] Mouse Cowpox in (small volume) Upper respiratory tract [52] Mouse Cowpox, vaccinia cut Skin lesions [53] Mouse (athymic) Cowpox, vaccinia cut Skin lesions [53,54] Mouse Vaccinia Skin scarif Skin lesions [55] (immunosuppressed) African dormouse Monkeypox ip Systemic [56] Ground squirrel Monkeypox ip Systemic [57,58] Rabbit Rabbitpox id Systemic [59] (animal-to-animal transmission) Rabbit Vaccinia WR strain id Systemic [59] Monkey Monkeypox itr Pneumonitis, skin lesions [60] Monkey Monkeypox iv Systemic, skin lesions [61] Monkey Variola iv Systemic, skin lesions [62] *Some skin and lung infections (pneumonitis) progressed to being systemic. The ectromelia infection models have been known for many years [10,11], but have only been recently exploited for antiviral studies. Mouse interleukin-4 (IL-4) producing virus, causing host immune dysfunction and severe disease. This reference describes dormice as an infected host; antiviral studies are in progress. CDV-R, cidofovir-resistant; cut, cutaneous; GFP, green fluorescent protein; id, intradermal; in, intranasal; inj, injection; ip, intraperitoneal; itr, intratracheal; iv, intravenous; scarif, scarification. virus infections. These include dormice (from Africa, now bred in the USA) and American ground squirrels (Table 2). These models arose following an outbreak of monkeypox virus in the USA acquired from the purchase of prairie dogs from a pet store that also sold exotic rodents from Africa [12]. Rabbits are being used more widely to study treatment of orthopoxvirus infections (Table 2). Rabbitpox virus causes a disseminated lethal infection, and the investigation of antiviral treatment on animal-to-animal transmission can be performed. Vaccinia (WR strain) was shown to cause disease in rabbits similar to that caused by rabbitpox virus, and might be equally useful to rabbitpox virus for chemotherapy studies. Besides aerosol infection, monkeypox virus models in monkeys now include those utilizing intratracheal and intravenous infection routes (Table 2). Chemotherapy of variola virus infections following intravenous infusion of virus in monkeys is also being investigated. These and other models are important to establish the efficacy of new antiviral compounds to satisfy the US Food and Drug Administration s animal efficacy rule for drug development against orthopoxvirus infections [13]. It is still unclear as to which animal models are most appropriate for predicting the activity of antiviral compounds against variola virus infections in humans. Antiviral compounds reported active against orthopoxviruses in animal models from The reader is referred to the review listing compounds that were found active in orthopoxvirus animal models for the years prior to 2003 [9]. During that time, compounds with the most notable potency and efficacy included CDV, cyclic CDV (cyclic HPMPC), S2242 and HOE-961 (the diacetate ester oral prodrug of S2242). The large number of other compounds reported active against orthopoxvirus infections in vivo did not compare in potency to the above four. After 2002, considerably more research has been conducted with CDV, but the other three compounds have been only Antiviral Chemistry & Chemotherapy

4 DF Smee minimally pursued. Many exciting new agents have been identified and are being actively pursued. These will be reviewed below. Activity of CDV in animal models since 2002 A number of experiments have been conducted using CDV to treat orthopoxvirus infections in various mouse models (Table 3). These include the newly employed ectromelia virus infection. CDV has been used to treat infections in newly developed models, such as upper respiratory infections induced by a low cowpox virus inoculum, infections with genetically altered viruses (ectromelia IL-4, cowpox-green fluorescent proteinproducing virus and CDV-R vaccinia virus) and cutaneous infections in hairless and athymic nude mice with cowpox and vaccinia viruses. In this research involving the treatment of cutaneous infections, topically Table 3. Activities of cidofovir in animal models of poxvirus infections since 2002 Animal species Virus Innoculation route Primary infection Effective treatment regimen Reference Mouse Ectromelia in Pneumonitis 5 mg/kg ip on day 0 and 1.25 mg/kg [63] on day 3 Mouse Ectromelia IL-4* Footpad inj Systemic 100 mg/kg/day ip daily starting 1 day [45] after infection; delayed but could not prevent death Mouse Cowpox, vaccinia in Pneumonitis mg/kg ip 1 from 5 days before [64] to 3 days after virus exposure Mouse, with cytokine Cowpox, vaccinia in Pneumonitis 100 mg/kg/day ip daily for 2 days [46] and chemokine starting 1 day after infection determinations Mouse, with cytokine Cowpox, vaccinia ip Systemic infection 100 mg/kg/day ip daily for 2 days [46] and chemokine starting 1 day after infection determinations Mouse (SCID) Cowpox, vaccinia ip Systemic infection 20 mg/kg/day ip daily for 7 days [64] starting 2 4 days after infection; delayed but could not prevent death Mouse Cowpox in Pneumonitis Aerosolized drug at estimated [65] mg/kg/day treated once starting 1 day before to 2 days after infection Mouse Cowpox Low volume in Upper respiratory 100 mg/kg/day ip daily for 2 days [52] tract infection starting 1 day after infection Mouse, with Cowpox-GFP ip Systemic infection 25 or 100 mg/kg ip 1 at 1 day [47] whole body imaging before infection Mouse (normal or Cowpox, vaccinia cut Skin lesions 50 mg/kg ip 1 /day or 1% or 5% [53,54] athymic nude) topical 1 /day or 3 /day starting 1 day after infection Mouse Vaccinia Skin scarif Skin lesions 100 mg/kg/day ip for 3 days or [55] (immunosuppressed) 1% topical 2 /day for 7 days starting 1 5 days after infection Mouse Vaccinia CDV-R in Pneumonitis 100 mg/kg/day ip on days 1 and 3 [48,49] non-lethal strains after infection; or 10 or 50 mg/kg/day ip for 5 days starting on the day of infection Mouse Vaccinia CDV-R in Pneumonitis 50 and 100 mg/kg/day ip 1 at 1 day [50] lethal strain after infection Mouse Vaccinia IHD strain in Pneumonitis 30 and 100 mg/kg/day ip 1 at [51] 1 day after infection Monkey Monkeypox itr Pneumonitis 5 mg/kg ip every other day for 5 or [60] 6 doses starting 1 day after infection Monkey Monkeypox iv Systemic infection 5 mg/kg iv given before or up to [66 68] 2 days after infection Monkey Variola iv Systemic infection 5 mg/kg iv given before or up to [66 68] 2 days after infection *Mouse interleukin-4 (IL-4) producing virus, causing host immune dysfunction and severe disease. CDV-R, cidofovir-resistant; cut, cutaneous; GFP, green fluorescent protein; in, intranasal; inj, injection; ip, intraperitoneal; itr, intratracheal; iv, intravenous; scarif, scarification; SCID, severe combined immunodeficient International Medical Press

5 Orthopoxvirus inhibitors in animals applied drug was more efficacious than parenteral administration. Further work with CDV in intranasal and intraperitoneal cowpox and vaccinia infections has been reported, including studies of the effects of CDV treatment on cytokines and chemokines produced during the infections. Because CDV is often the positive control in orthopoxvirus studies in mice, it will most likely continue to be used for future experiments. Studies were conducted using CDV to treat monkeypox and variola virus infections in monkeys (Table 3). Treatment with the drug led to considerable reductions in lesion number over the body of the animal, decreases in tissue virus titres and prevention of death. Activities of ether lipid prodrugs of CDV and (S)-HPMPA in animal models Although CDV is highly active against orthopoxviruses in animal models, it is not orally bioavailable and causes renal toxicity [14]. Investigators have discovered ether lipid prodrugs of CDV that address both problems: the compounds are orally active and exhibit reduced nephrotoxicity [15]. Four such compounds, HDP-CDV, ODBG-CDV, ODE-CDV and OLE-CDV, have all shown activity against ectromelia and/or cowpox and vaccinia virus respiratory infections in mice (Table 4). The compound that has been most studied is HDP-CDV. HDP- CDV and the three other compounds are more potent than CDV in mouse models. HDP-CDV was also found to be active against rabbitpox virus infection in rabbits (Table 4). This compound is being actively pursued for clinical development for the oral treatment of orthopoxvirus infections. (S)-HPMPA is the adenine equivalent to CDV (formerly referred to as (S)-HPMPC, and containing cytidine). Investigators found that HDP-HPMPA and ODE-HPMPA have properties similar to their counterpart compounds, HDP-CDV and ODE-CDV, in the Table 4. Activities of ether lipid prodrugs of (S)-HPMPA and cidofovir in animal models of poxvirus infections since 2002 Animal Innoculation Primary Compound species Virus route infection Effective treatment regimen References HDP- Mouse Cowpox, vaccinia in Pneumonitis mg/kg/day po once daily for 5 days [69] HPMPA starting 1 2 days after infection ODE- Mouse Cowpox, vaccinia in Pneumonitis mg/kg/day po once daily for 5 days [69] HPMPA starting 1 2 days after infection HDP-CDV Mouse Ectromelia Aerosol Pneumonitis 5 10 mg/kg/day po for 5 days starting 4 h [42,63,70] after infection; or 5 mg/kg on day 0 and 25 mg/kg on day 3 HDV-CDV Mouse Ectromelia in Pneumonitis 10 mg/kg po on day 1 of infection [63] and 25 mg/kg every other day thereafter starting up to 5 days after infection HDV-CDV Mouse Cowpox, vaccinia in Pneumonitis 5 7 mg/kg/day po for 5 days starting 1 3 days [71] after infection, or 12 mg/kg po 1 at 1 3 days after infection HDV-CDV Mouse Cowpox in Pneumonitis mg/kg/day po in combination with [72] ST-246 (1 10 mg/kg/day po) HDV-CDV Mouse Vaccinia CDV-R in Pneumonitis mg/kg/day po 1 at 1 day after infection [50] lethal strain HDV-CDV Mouse Vaccinia IHD strain in Pneumonitis mg/kg/day po 1 at 1 day after infection [51] HDV-CDV Rabbit Rabbitpox id Systemic 5 mg/kg/day po twice daily for 5 days starting 1 [59] infection day before infection ODBG- Mouse Ectromelia in or aerosol Pneumonitis 2 8 mg/kg/day po for 5 days starting 4 h after [70] CDV infection ODE-CDV Mouse Ectromelia Aerosol Pneumonitis mg/kg/day po for 5 days starting 4 h [42] after infection ODE-CDV Mouse Cowpox, vaccinia in Pneumonitis 5 7 mg/kg/day po for 5 days starting 1 2 days [71] after infection or 10 mg/kg po 1x at 1 3 days after infection OLE-CDV Mouse Ectromelia Aerosol Pneumonitis 3 mg/kg/day po for 5 days starting 4 h [42] after infection ODE-CDV Mouse Cowpox, vaccinia in Pneumonitis 5 7 mg/kg/day po for 5 days starting [71] 1 2 days after infection CDV, cidofovir; CDV-R, cidofovir-resistant; id, intradermal; in, intranasal; po, peroral. Antiviral Chemistry & Chemotherapy

6 DF Smee Table 5. Activities of ST 246 in animal models of poxvirus infections since 2002 Animal species Virus Innoculation route Primary infection Effective treatment regimen References Mouse Ectromelia in Pneumonitis mg/kg/day po once or twice daily for [16,73] 10 or 14 days starting up to 3 days after infection Mouse Cowpox, vaccinia in Pneumonitis mg/kg/day po 1 to 3 daily for 7 14 days [16,73] starting 1 2 days after infection Mouse Cowpox in Pneumonitis 1 10 mg/kg/day po in combination with HDP-CDV [72] (0.3 3 mg/kg/day po) starting 1 or 3 days after infection Mouse Vaccinia iv Tail lesions mg/kg/day po twice daily for 5 days [16] starting 2 h after infection Rabbit Rabbitpox Aerosol Pneumonitis 40 mg/kg/day po once daily for 14 days starting [74] up to 3 days after infection Ground squirrel Monkeypox sc Systemic 100 mg/kg/day po for 14 days starting [58] 1 4 days after infection Monkey Monkeypox iv Systemic 300 mg/kg/day po once daily for 14 days starting [75] 1 or 3 days after infection Monkey Variola iv Systemic 300 mg/kg/day po once daily for 14 days starting [75] 1 or 3 days after infection CDV, cidofovir; in, intranasal; iv, intravenous; po, peroral; sc, subcutaneous. inhibition of cowpox and vaccinia intranasal infections in mice (Table 4). These adenine compounds are orally active against the infections and non-toxic, differing from their parent compound (S)-HPMPA. Activity of ST 246 in animal models ST-246 is perhaps the most interesting and important newly discovered anti-orthopoxvirus agent reported since The compound is not a nucleoside analogue like the CDV and (S)-HPMPA series, and therefore does not inhibit viral DNA synthesis. Instead, ST 246 blocks a late step in virus assembly preventing intracellular envelope virus formation and subsequent virus egress from the cell [16]. Thus, the spread of virus in vivo is greatly impaired. ST 246 has shown efficacy in the treatment of ectromelia, cowpox and vaccinia virus infections in mouse models, and rabbitpox virus infections in rabbits (Table 5). ST 246 was effective, in combination with HDP-CDV, in treating cowpox virus respiratory infections in mice. The compound has shown efficacy against monkeypox virus infections in ground squirrels, and against monkeypox and variola virus infections in monkeys (Table 5). The dose used to treat monkeypox and variola virus infections in monkeys was high. However, doses as low as 3 mg/kg/day are also very effective (Robert Jordan, Siga Technologies, personal communication). Treatment of infections in animals requires a considerably longer course (10 14 days) than other agents, such as CDV (1 2 days using high doses), but the benefits are great. Importantly, ST 246 was used to treat a child with a disseminated vaccinia virus infection, who dramatically improved after apparent failure from prior treatment with CDV and vaccinia immune globulin [17]. Recently the results of a Phase I clinical trial of ST 246 in humans were published [18]. The compound was well tolerated at single oral doses as high as 2 g in fasting volunteers and 1 g in non-fasting volunteers. These and lower doses were well tolerated. The exposure levels were predicted to be adequate for the treatment of orthopoxvirus infections in man. Activities of various other compounds in animal models A number of unrelated compounds have been tested in mouse infection models since 2002 (Table 6), with varying degrees of potency. Adenine arabinoside (Ara A) and FMAU were active against a cowpox virus respiratory infection in mice. The potency of Ara A is low, and FMAU, although effective, is very closely related to fialuridine (FIAU), a lethally toxic compound to humans [19]. CI 1033, Gleevec TM, HOE-961 (prodrug of S2242), isatin-β-thiosemicarbazone, interferon-α, interferon-γ, marboran and N-methanocarbathymidine (N MCT) were active against a vaccinia virus respiratory infection in mice. CI 1033 and Gleevec TM are interesting because they disrupt a cellular signal transduction process (via tyrosine kinase inhibition [20,21]) important for virus replication, rather than inhibiting a virus-specific enzyme. This prevents extracellular envelope formation and release. Importantly, isatin-β-thiosemicarbazone and marboran were not effective against cowpox virus infections in mice [22], thus dampening the enthusiasm for these inhibitors. Although N MCT was effective in treating mice both orally and parenterally, the compound is considerably more potent in infected mouse cells compared with rabbit, monkey and human cells International Medical Press

7 Orthopoxvirus inhibitors in animals [23], suggesting that it might prove to be less active if tested in rabbit and monkey models, or in humans. S2242 was highly effective in treating a CDV-R vaccinia virus infection in mice. S2242 has not shown sufficient activity against variola virus in cell culture (John Huggins, USAMRIID, personal communication) and is therefore not being pursued. A recombinant vaccinia virus encoding for interferon-λ was inhibited in its replication in mice, implicating interferon-λ as an anti-poxvirus protein (Table 6). A newly synthesized nucleoside phosphonate, HPMPO-DAPy, was effective in tail lesion and cutaneous infections in mice and in treating monkeypox virus-induced pneumonitis in monkeys. It remains to be seen whether this compound will be further developed. Recently, the efficacy of an orally active 4 thio derivative of iododeoxyuridine, SRI 21950, was described. This compound is highly potent in mice infected with cowpox and vaccinia viruses over a broad range of doses, and deserves further investigation in other animal models. Summary of progress made A number of newly developed or refined mouse, African dormouse, ground squirrel, rabbit and monkey models are available for studying the treatment of orthopoxvirus infections. Investigations have led to the discovery Table 6. Activities of other compounds in animal models of poxvirus infections since 2002 Innoculation Primary Compound Animal species Virus route infection Effective treatment regimen References Ara-A Mouse Cowpox in Pneumonitis 300 mg/kg/day ip twice daily for 5 days starting [76] 1 day after infection CI-1033 Mouse Vaccinia in Pneumonitis 50 mg/kg/day ip once daily for 8 days starting 6 h [20] prior to infection FMAU Mouse Cowpox in Pneumonitis 100 mg/kg/day ip twice daily for 5 days starting [76] 1 day after infection Gleevec (ST-571) Mouse Vaccinia in Pneumonitis 100 mg/kg/day by sc Alzet pump starting 1 day [21] prior to infection HOE-961 (prodrug Mouse Vaccinia in Pneumonitis mg/kg/day po twice daily for 5 days [51] of S2242) IHD strain starting 1 day after infection Isatin-β- Mouse Vaccinia in Pneumonitis mg/kg/day ip once daily for 5 days starting [22] thiosemicarbazone 1 or 2 days after infection HPMPO-DAPy Mouse Vaccinia iv Tail lesions mg/kg/day ip once daily for 5 days starting [77] 2 h after infection Mouse Vaccinia cut Skin lesions 100 mg/kg/day sc on days after infection [78] (athymic nude) Monkey Monkeypox itr Pneumonitis 5 mg/kg ip every other day for 5 or 6 doses [60] starting 1 day after infection Interferon-α Mouse Vaccinia in Pneumonitis 5,000 U/day in once daily for 5 days starting 1 day [79] before infection Interferon-γ Mouse Vaccinia in Pneumonitis 2,000 U/day in once daily for 5 days starting 1 day [79] before to 2 days after infection Interferon-λ Mouse Vaccinia in Pneumonitis Genetically modified virus expressing [80] interferon-λ during infection Mouse Vaccinia id Skin lesions Genetically-modified virus expressing [80] interferon-λ during infection Marboran Mouse Vaccinia in Pneumonitis mg/kg/days ip once daily for 5 days starting [22] 1 3 days after infection N-MCT Mouse Cowpox, in Pneumonitis mg/kg/day ip or po at mg/kg/day [23,81,82] vaccinia for 5 days starting 1 day after infection S2242 Mouse Vaccinia, in Pneumonitis mg/kg/day ip twice daily for 5 days starting [50] CDV-R lethal 1 day after infection strain SRI (4 -thio Mouse Cowpox, in Pneumonitis mg/kg/day ip twice daily for 5 days starting [83] derivative of iodo- vaccinia 1 3 days after infection deoxyuridine) Ara-A, adenine arabinoside; CDV-R, cidofovir-resistant; cut, cutaneous; id, intradermal; in, intranasal; ip, intraperitoneal; itr, intratracheal; iv, intravenous; N-MCT, N methanocarbathymidine; sc, subcutaneous. Antiviral Chemistry & Chemotherapy

8 DF Smee of orally active inhibitors of orthopoxvirus infections in animals, including ether lipid prodrugs of CDV and (S)-HPMPA, ST 246, N MCT and SRI Another promising acyclic nucleoside phosphonate compound is HPMPO-DAPy. With this list of new inhibitors to work with, the goal of having at least two compounds available for the treatment of orthopoxvirus infections in humans appears to be within reach. Acknowledgements This work was supported by Contract N01-AI from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The article is dedicated to my mentor and friend, Dr Robert W Sidwell, who co authored the 2003 publication [9]. He retired in November 2006 as Founder and Director of the Utah State University Institute for Antiviral Research. Disclosure Statement The author declares no competing interests. References 1. Breman JG, Henderson DA. Poxvirus dilemmas monkeypox, smallpox and biological terrorism. 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