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6 Development of Therapeutics H istorical evidence suggests that, in response to an accidental or intentional release of variola virus, smallpox vaccination would be an effective public health measure to protect at-risk populations. To be protective, however, vaccination must occur within 4 days of exposure to the virus (Fenner et al., 1998; Mortimer, 2003). In addition, there are contraindications to the administration of current smallpox vaccines, par- ticularly among immunocompromised individuals. These individuals would need alternative protective measures following exposure to variola virus. To reduce the significant morbidity and mortality in cases of smallpox, safe and effective therapeutic agents are required. By accelerating clearance of the virus from ill individuals, such agents may also limit infectivity and transmission of disease. Antiviral agents can also be useful for prophylaxis after exposure has occurred. The availability of these agents has the poten- tial to be important for both the treatment and prophylaxis of smallpox in exposed persons identified after the 4-day period when vaccination is effec- tive, and could be a valuable component of any effective control strategy. In the last decade, substantial progress has been made in the development of therapeutics with the potential to meet this need (see Tables 6-1 and 6-2). However, these efforts have yet to yield an FDA-licensed agent for the treat- ment or prevention of smallpox and other orthopoxviruses. The 1999 IOM report identified the development of antiviral agents as the most significant reason to retain stocks of live variola virus, primarily because of the lack of availability of an effective therapeutic agent (either currently or historically) that could serve as a standard for purposes of comparison: 

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 LIVE VARIOLA VIRUS The most compelling reason for long-term retention of live variola virus stocks is their essential role in the identification and develop- ment of antiviral agents for use in anticipation of a large outbreak of smallpox. It must be emphasized that if the search for antiviral agents with activity against live variola virus were to be continued, additional public resources would be needed. The 1999 report also suggested that having more than one antiviral agent would be desirable because of the potential for the emergence of drug-resistant variola strains. Replication-deficient forms of variola virus could be used to develop new agents; ultimately, however, the live intact virus would be required to ensure confidence in the results. The 1999 report also noted that, given the lack of incentive for the development of smallpox therapeutics in the private sector, significant public resources would need to be mobilized. This chapter reviews potential therapeutics for smallpox, regulatory requirements for the development of such therapeutics, and the need for live variola virus in this work. POTENTIAL THERAPEUTICS FOR SMALLPOX Potential therapeutics for smallpox include two drugs approved by the FDA for other purposes, newly developed drugs, agents to block newly identified poxvirus targets, and drugs that enhance or modulate the host’s immune response. Use of Drugs Approved by the FDA for Other Purposes Because de novo drug development is an expensive and time-consuming process (costing in excess of $500 million and requiring approximately 8–10 years of continuous effort) (Henderson and Fenner, 2001), the use of licensed drugs approved for other purposes represents an attractive option for antivirals against variola. Cidofovir is a DNA polymerase inhibitor, licensed for the treatment of cytomegalovirus-induced retinitis in HIV-infected individuals (Tesh et al., 2004). Cidofovir also exhibits in vitro antiviral activity against poxviruses, and is effective against cowpox and vaccinia virus infections in mice (LeDuc et al., 2002; Baker et al., 2003; Quenelle et al., 2003; Magee et al., 2005). Under an Investigational New Drug (IND) protocol from the FDA, cidofovir can be used to treat acute smallpox and complications arising from vaccinia infection when a patient has not responded to administra- tion of vaccinia immune globulin (VIG) (LeDuc et al., 2002; reviewed in Sliva and Schnierle, 2007). However, the utility of cidofovir for treating

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 DEVELOPMENT OF THERAPEUTICS smallpox is complicated by the fact that the drug is available only in a topical or intravenous formulation. A topical formulation would have no role in treating a systemic disease such as smallpox. Intravenous cidofovir must be given as a 1-hour infusion in combination with multiple doses of probenecid and requires sustained intravenous hydration and monitor- ing of renal function. Even when given intravenously, the drug does not cross the blood–brain barrier. Although cidofovir’s long half-life has the advantage of allowing weekly dosing, problems with administration and toxicity make large-scale use of this agent difficult. It is not likely to be usable in resource-poor settings. The emergence of resistance is also a con- cern because exposure of vaccinia to cidofovir resulted in the emergence of mutations in the DNA polymerase gene, which is the target of the drug (Becker et al., 2008). Gleevec (also referred to as STI-571 or imatinib mesylate) is an FDA- approved treatment for chronic myeloid leukemia that exhibits antiviral activity against poxviruses. Gleevec blocks the action of Abl-family tyrosine kinases (Druker et al., 1996) and thus blocks the egress of vaccinia virus from infected cells in vitro (McFadden, 2005; Reeves et al., 2005; Yang et al., 2005). It has also undergone in vitro testing against the monkeypox and variola viruses with similar effects (Reeves et al., 2006). In addition, Gleevec treatment promoted survival of mice following intranasal challenge with vaccinia virus, and it has been suggested as a potential therapeutic for postvaccination complications associated with vaccinia (Reeves et al., 2005). The drug does not appear to interfere with the development of immunity that protects against subsequent challenge. However, the protective benefit of Gleevec was evident only at lower virus titers and only when the drug was given less than 48 hours after exposure. Studies of Gleevec in rabbits infected with rabbitpox and in mice infected with ectromelia showed much lower antiviral activity than in other animal models (personal communication, Dr. Daniel Kalman, Emory University, February 2009). The reduced activity against higher titers of the inoculum virus, the requirement for administra- tion shortly after inoculation, and the variable protection in poxvirus models raise concerns about Gleevec’s potential for treating smallpox. Newly Developed Therapeutics To overcome the challenges associated with cidofovir discussed above, orally bioavailable cidofovir derivatives have recently been developed (HDP-cidofovir/CMX-001) (Ciesla et al., 2003; Buller et al., 2004; Kern et al., 2004). CMX-001 also displays enhanced antiviral activity against variola virus in comparison with cidofovir (Bradbury, 2002; Morris, 2002; Sliva and Schnierle, 2007). The inhibitory activity of hexadecyloxypropyl- CDV is 40–100 times grater than that of CDV in vitro in cells infected with

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TABLE 6-1 Development of Therapeutics for Smallpox 0 Drug Chemical Structure Mode of Action Reference Cidofovira ATP analog that inhibits Baker et al., 2003; Magee et al., NH2 (CDV) DNA polymerase. When 2005, 2008; Krecmerová et al., incorporated into the 2007a N X template strand, blocks DNA elongation and 3′–5′ proof- Y N O reading exonuclease activity. Drug resistance seen in the VV E9L gene (DNAP). FDA OH approved for cytomegalovirus O P(O)(OH)2 retinitis in persons with HIV. 1, X = N, Y = CH 2, X = CH, Y = N CDV CMX-001 Same target as CDV. Kern et al., 2002; Kern, 2003; derivativesb,c: Esterification makes these Krecmerová et al., 2007b; Quenelle NH2 CMX001 and derivatives more lipophilic et al., 2007b; Magee et al., 2008; Table 6-1, Cidofovir (CDV) HPMP-5-azaCb and increases uptake roughly Naesens et al., 2008; Parker et al., N 50-fold. CMX001 is 100-fold 2008 R01478 more active than CDV and aw NO does not produce renal redrO n with vectors toxicity. O P OCH2CH2CH2OCH2(CH2) 14CH3 O- Na+ HO Table 6-1, CMX001 R01478

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vector, editable HPMP-5-azaCb NH2 N N O N O O PO OR STI-571 Blocks the Abl-family McFadden, 2005; Reeves et al., 2005 N (Gleevec or tyrosine kinases needed for imatinib the actin motility of N HN N mesylate) intracellular viral particles Table 6-1, HPMP-5-azaC (IMV), thus blocking egress CH3 of IMV from cells. FDA R01478 approved for chronic myeloid redrawn as vectors leukemia. HN CH3 O N N Table 6-1, STI-571 R01478  continued redrawn as vector

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TABLE 6-1 Continued  Drug Chemical Structure Mode of Action Reference ST-246d Inhibits virus release by Yang et al., 2005; Quenelle et al., H targeting a pox protein (p37 2007a,b HH or 60L for cowpox or F13L H for vaccinia) that is essential O for envelopment of IMV. N O O HN F F F Table 6-1, ST-246 R01478 redrawn

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Targets the Thymidine Kinase Kern et al., 2009 4′-thioIDUe O (TK) gene to inhibit DNA R synthesis. NH N O YO SX OY aCidofovir {HPMPC, CDV, 1-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl] cytosine}. b{1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine; HPMP-5-azaC} is an analog of CDV. cCMX001 is hexadecyloxypropyl CDV and has much better oral bioavailability than CDV. dST-246 [N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-di oxo-4,6-etheneocycloprop[f]isoindol-2(1H)-yl)-benzamide] used at 20 µg/ml in cell cultures re- duces EMV by 6 logs and reduces IMV by 2 logs. e1-(2′-deoxy-4′le 6-1, 4’-thioIDU Tab-thio-β-D-ribofuranosyl)-5-iodouracil (4′-thioIDU). The R can be F, Br, I, CH3, CF3, or a phenyl group. The X can be H or OH. The Y can be H or an acetyl group. R01478 redrawn as vectors 

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 LIVE VARIOLA VIRUS TABLE 6-2 Clinical Aspects of Therapeutic Agents for Smallpox in Humans Dose and Route of Drug Administration Issues Cidofovir 5 mg/kg given intravenously Severe renal toxicity; co-administration of weekly/biweekly; also used probenecid necessary; hydration topically and intralesionally requirement; used off label for recurrent laryngeal papillomatosis, molluscum contagiosum, and human papillomavirus Esters of Dose not yet defined; taken Phase I and II human clinical trials under Cidofovir orally way; no reported renal toxicity to date; (CMX001) higher bioavailability than CDV Gleevec 400–800 mg/day; taken Chemotherapeutic agent; side effects include orally edema, cytopenia, and hepatotoxicity ST-246 500–2000 mg/day; taken Minimal toxicity seen in human dosing orally for 14 days; other trials routes of administration (intravenous, liquid suspension) being considered variola, cowpox, vaccinia, or ectromelia virus. Protection of mice from lethal mousepox infection has been demonstrated (Parker et al., 2008), and CMX-001 was effective against mousepox in the C57BL/6 strain, which is considered to have a course of infection more similar to that of variola than its progression in other mousepox strains when given 4 days after inoculation (Parker et al., 2009). Other derivatives of cidofovir could prove effective as well (Lebeau et al., 2006; Stittelaar et al., 2006; Hostetler et al., 2007; Hostetler, 2009). Orally bioavailable cidofovir derivatives have shown negligible renal toxicity, a significant advantage over the intravenous formulation. CMX-001 has been given to a patient with eczema vaccinatum who did not respond to ST-246 (CDC, 2009). A recently completed human volunteer phase I multidose study with more than 100 subjects demon- strated no significant adverse events, and phase II trials are being initiated (Painter and Hostetler, 2004; Ruiz et al., 2007). ST-246, which was discovered from a high-throughput screen of 356,240 small-molecule inhibitors of vaccinia virus replication, is currently being used in human trials. This antiviral drug targets the vaccinia virus protein F13, which is essential for envelopment and egress of the intracellular mature virions (MV) and subsequent viral spread (Yang et al., 2005). Cell cultures infected with six different variola isolates or seven different monkey- pox isolates showed reduced cytopathic effects, virus production, and comet

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 DEVELOPMENT OF THERAPEUTICS tail formation after treatment with nanomolar amounts of ST-246. ST-246 is 8,000 times more potent than cidofovir in vitro against poxviruses, is orally bioavailable, and is stable at room temperature. It has proven to be effective in blocking replication of all orthopoxviruses that have been tested in vitro (Duraffour et al., 2007) and in protecting mice (Yang et al., 2005; Quenelle et al., 2007a), rabbits (Nalca et al., 2008), and ground squirrels (Sbrana et al., 2007) from orthopoxvirus challenge. Animals infected with monkeypox, cowpox, ectromelia, and variola viruses that received ST-246 were protected from lethal infection and also mounted a protective immune response (Bolken and Hruby, 2008; Nalca et al., 2008). ST-246 in combina- tion with CMX-001 displays synergistic antiviral effects against vaccinia and cowpox in animals without increasing toxicity (Quenelle et al., 2007b; Whitley, 2008). In 2007, a 14-day course of ST-246 was used in conjunction with cidofovir and VIG under an emergency IND to treat a severe case of eczema vaccinatum in an infant who was infected with vaccinia as a result of contact transmission (Vora et al., 2008). Since cidofovir and VIG were co- administered with ST-246, however, it is not clear that the resolution of the infection is attributable entirely or even partially to ST-246. Human phase I trials of ST-246 have been completed. The drug was given to 31 healthy individuals in a single dose ranging from 500 mg to 2000 mg daily in a fasting and nonfasting state, with an 8-person placebo group used for com- parison (Jordan et al., 2008). Side effects were minimal, and only reversible neutropenia was seen more often in the treated than in the placebo group. Important information on ST-246 has been obtained: the variola gene product targeted by ST-246 is known, and the doses have been shown to be effective against poxviruses in mice and nonhuman primates. However, clinical data are needed on the use of ST-246 in humans; studies to provide these data are under development for naturally occurring human monkey- pox but will be difficult to implement and monitor. An important caveat for antiviral drugs such as ST-246 that exhibit high potency in vitro is that they can be tested only in model systems or against other poxvirus infections in humans, and it is impossible to know with certainty how they would perform against smallpox in the event of its reemergence. Work on the development of new drugs has also continued in Russia. VECTOR reports having conducted screening of more than 5,000 chemi- cal compounds for their antiviral activity, and about 80 compounds active against surrogate orthopoxviruses (vaccinia virus, cowpox virus, and ectromelia virus) are said to have been identified. In testing done in cell culture, VECTOR reports that 60 compounds demonstrated antiviral activity against variola virus (Zakirova et al., 2004; Ivanov et al., 2005, 2008; personal communication, Ilya Drozdov, WHOCC for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and DNA, March 27, 2009).

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 LIVE VARIOLA VIRUS Agents to Block Newly Identified Poxvirus Targets Further research on poxvirus replication has made it possible to identify possible poxvirus drug targets, and assessment of molecules blocking these targets has begun (Yang et al., 2005; Sliva and Schnierle, 2007; Tse-Dinh, 2008). Three enzymes involved in vaccinia virus replication have been iden- tified and crystallized: thymidine kinase (TK), deoxyuridine triphosphatase (DUTPase), and uracil DNA glycosylase (UDG) (Whitley, 2008). DNA polymerase nucleoside inhibitors (Fan et al., 2006; Prichard et al., 2007), nucleoside inhibitors of S-adenosyl-L-homocysteine hydrolase (De Clerq and Holy, 2005; Roy et al., 2005; Yang and Schneller, 2005; Arumugham et al., 2006), targets of topoisomerase I (Da Fonseca and Moss, 2003; Bond et al., 2006; Fujimoto et al., 2006; Perry et al., 2006), and other egress inhibitors (Bailey et al., 2007) have been evaluated for their ability to block poxvirus replication. It has been suggested that the new 4′ thioIDU (TK inhibitor) might be an additional component of combination therapy since it can block replication of CMX–001- and ST-246-resistant mutants (Kern et al., 2009). The use of combination antiviral therapy is favored as it may slow the development of drug-resistant strains of variola and other orthopoxviruses. When administered intraperitoneally or orally, 4′ thioIDU was shown to be protective against both cowpox and vaccinia in mice (Kern et al., 2009). Selectivity indices (CC50/EC50) ranged from more than 200 to 2,000 for 4′ thioIDU; in contrast, the values for CDV were more than 9 to more than 32 (Kern et al., 2009). However, 4′ thioIDU, like CDV and its derivatives, is toxic for dividing cells. Agents That Enhance or Modulate the Host Immune Response Enhancing or modulating the host immune response is an alterna- tive or adjunctive therapeutic approach to controlling smallpox through antiviral drugs that disrupt the replication cycle. Providing passive immu- nity through the transfer of protective antibodies from an immune to a susceptible individual can lend temporary, but potentially life-saving, protection. As an example, this approach was used therapeutically in the 1940s in Morocco. Antiserum was obtained from smallpox survivors soon after the last scabs fell off, and was then administered to newly arriving patients at the clinic in doses of 10–20 ml per day (Couzi and Kircher, 1941). Among the 200 persons given this treatment, including 75 patients with advanced hemorrhagic disease, all survived. However, this was a report of clinical experience, not a controlled study, and use of passive antibodies as therapy for clinically evident, established infection has not been demonstrated to be effective against systemic viral illnesses.

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 DEVELOPMENT OF THERAPEUTICS Today, VIG collected from individuals with high antibody titers from repeated immunization is given to confer passive immunity in individuals with complications resulting from smallpox vaccination, and is the only cur- rently available intervention other than unlicensed antiviral drugs (Kempe et al., 1961; Wittek, 2006). Two intravenous formulations of VIG (Cangene and Dynport) have been licensed by the FDA for the management of patients with progressive vaccinia, eczema vaccinatum, severe generalized vaccinia, and extensive body surface involvement or periocular implanta- tion of vaccinia following inadvertent inoculation (Wittek, 2006). When given to exposed individuals, VIG is expected to provide protection against infection for approximately 2–3 weeks, presumably through its neutralizing activity against vaccinia. The conserved orthopox protein vaccinia B5/variola B6 is a major neutralizing target for VIG, although major neutralizing sites on B5 are exposed differently on the variola ortholog (Aldaz-Carroll et al., 2007). B5 is needed to wrap the MV to form extracellular virus, and interactions with actin are necessary for virion egress from the infected cell (see Aldaz-Carroll et al., 2005, 2007). More recently, humanized chimpanzee monoclonal antibodies specific for the B5 and A33 envelope glycoproteins of vaccinia virus and the variola virus homologs have been reported to inhibit the spread of vaccinia and variola viruses in vitro and have conferred protection in a mouse model of poxvirus infection (Chen and Ron, 2006; Chen et al., 2007). These antibodies may be useful for treating vaccine-related complications or for prophylaxis or therapy of smallpox. VECTOR reports that since 2002 it has been working to develop human recombinant antibodies as therapeutics for treatment of smallpox infection (Tikunova et al., 2005; Yun et al., 2006; Dubrovskaia et al., 2007). To that end, a panel of 66 unique human mini-antibodies against orthopoxviruses, including variola virus, was selected from VECTOR’s combinatory phage library and from that obtained from The Medical Research Council (UK). Half of the antibodies selected were tested for their ability to neutralize variola virus. Based on the most promising antibodies, VECTOR states that four fully human antibodies against variola virus were constructed, their affinity constants were measured, and they were tested for their ability to neutralize vaccinia virus (personal communication, Ilya Drozdov, WHOCC for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and DNA, March 27, 2009). Other antiviral drugs, such as ribavirin, that are not poxvirus specific but counteract host responses represent another therapeutic approach to smallpox infection (Baker et al., 2003). The lower specificity and poten- tial toxicity of such drugs make them less ideal, but some have been powerful modulators of disease severity with life-saving effects. Two recent

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 LIVE VARIOLA VIRUS reviews have suggested that agents such as tumor necrosis factor (TNF) inhibitors that are used to treat septic shock may be effective (Harrison et al., 2004; Jahrling et al., 2005). However, recent transcriptome profiling studies (Rubins et al., 2004) have shown that virulent poxvirus infection in primates appears to suppress TNF expression, raising concerns that further TNF suppression may enhance virulence and produce more severe disease. Moreover, several laboratory studies have suggested that the TNF- inhibiting genes of the poxviruses are a crucial part of pathogenesis (Sedger et al., 2006; Bartee et al., 2009). TNF production therefore is likely to be a protective mechanism counteracted by orthopoxvirus proteins. This finding also suggests that disease would be exacerbated by anti-TNF treatment. As noted, postexposure smallpox vaccination is beneficial if given shortly after the contact. In addition to accelerating the development of specific antiviral responses, vaccinia inoculation may elicit immediate innate responses that control the initial progression of infection and modulate disease severity. In a monkeypox model, however, postexposure vaccination was not as effective as cidofovir or its derivatives (Stittelaar et al., 2006). Summary At present, two drugs that are FDA-approved for other purposes— cidofovir, a DNA synthesis inhibitor, and Gleevec, a tyrosine kinase inhibitor—hold potential for use as therapeutics against smallpox. Cidofovir can be used on an investigational basis for treating severe orthopoxvirus infections, including smallpox. FDA-approved preparations of VIG are also available. New drugs that are under evaluation and show promise include orally bioavailable esters of cidofovir (CMX-001) and ST-246, an inhibitor of virus egress. ST-246 has been given to human volunteers and has been administered on a compassionate use therapeutic basis to a 2-year-old child with eczema vaccinatum following vaccinia exposure. New types of VIG are also being developed that target specific proteins such as variola B6R. REgULATORy REqUIREMENTS Recommendations and requirements for U.S. licensure of drugs intended for the prevention and treatment of variola infection are outlined exten- sively in a 2007 Guidance for Industry document prepared by the FDA (see also Chapter 1) (FDA, 2007). The guidance pertains primarily to small- molecule therapeutics, although its main principles can also be applied to biological products such as immunoglobulin preparations, monoclonal anti- bodies, and therapeutic proteins. Of particular relevance, demonstration of efficacy against live variola virus appears to be an essential step on the pathway to licensure (see Table 6-3). More specifically, use of the Animal

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 DEVELOPMENT OF THERAPEUTICS TABLE 6-3 Scientific Pathway for Drug Development Steps Assays Criteria 1. Rational Design Computerized displays of viral Drug fits target (optional) proteins and best fit of drugs 2. Cell culture tests Drug effects on cytotoxicity, Efficacy/toxicity (EC50/CC50) of effects of drugs virus production, cytopathy, >10 on infected cells comet formation, generation of resistant mutants 3. Small-animal Use of mice infected with Doses and routes for treatment model ectromelia, cowpox, or found where virus titers decrease vaccinia for initial studies of by >3 logs, disease signs are drug safety and efficacy in vivo eliminated in most animals, and mortality decreases >50% 4. Large-animal Cynomolgus macaques given Same as above model monkeypox intratracheally or variola intravenously should be tested for shedding, virus titers, disease signs or A nonrodent model, such as one using rabbits or monkeys, should be tested for shedding, virus titers, transmission, disease signs • Phase I/II clinical trials 5. Human beings Safety trials in humans should • Treatment or emergency use monitor blood chemistries and other biomarkers for toxicity; Investigational New Drug infected people should also be (IND) application for severe tested for virus vaccinia or other orthopoxvirus infections • Emergency Use Authorization (see Chapter 1) Rule (see Chapter 1) or any other currently available regulatory pathway to achieve licensure is essentially precluded by the exceptionally narrow host range of variola virus; the lack of any previously recognized effective drug for use in head-to-head comparison with any new compound; and known and possible differences between variola and other orthopoxviruses in dis- ease characteristics, drug susceptibility, and host range (Jordan and Hruby, 2006; Bolken and Hruby, 2008). Further, FDA officials have highlighted the

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0 LIVE VARIOLA VIRUS critical importance of conducting safety studies in normal human volunteers and potentially in patients with underlying medical conditions. The FDA also recommends studies using animal models that mimic human disease progression to provide supporting evidence of clinical efficacy (Roberts et al., 2008), but the development of a nonhuman primate challenge model for variola has been extraordinarily difficult in practice (see Chapter 4); more- over, some orally administered candidates (e.g., CMX-001) are not absorbed in these animals. While data derived from studies of other orthopoxviruses (e.g., monkeypox or vaccinia) cannot be considered definitive evidence of antivariola activity, the FDA guidance indicates that exploratory studies with these viruses can provide important adjunctive information. In addition to variola-specific considerations, general considerations applicable to the licensure of any antiviral agent include analysis of in vitro activity in conjunction with other drug candidates, selection and evaluation of resistant viral strains, and consideration of drug–vaccine and drug–drug interactions. NEED FOR LIVE VARIOLA VIRUS Fewer than 10 percent of published studies related to the development of therapeutics for smallpox have actually involved the use of live variola virus. This fact demonstrates that much can be accomplished by other means. For both scientific and regulatory reasons, however, the advanced stages of drug development will require evaluations involving live variola virus. In the 30 years since the eradication of smallpox, variola stocks have been used to complete the sequence of at least 49 VARV isolates (see Chap- ter 5) (Esposito et al., 2006), to gain some understanding of the genetic differences between virulent and nonvirulent poxviruses, to understand the neutralizing epitopes that could be targeted by VIG, to further understand the replication cycle of variola in order to identify potential targets for antiviral agents, and to design and evaluate potential variola model chal- lenge systems for purposes of confirming the efficacy of candidate antiviral agents under the Animal Rule. Although preliminary testing of antivirals can use related orthopox- viruses, live variola virus should be used in cell culture as the ultimate test. Host cell responses that define the course of variola infection in cell cul- ture or in vivo, such as changes in gene expression or changes in signaling pathways, miRNA, or secreted cytokines, should be investigated to identify networks of responses that could serve as biomarkers of inhibition of virus infection by a candidate drug. Host responses to similar viruses, such as monkeypox or vaccinia, could be used to identify biomarkers associated with virulent or benign infection. The changes in these profiles found to be associated with successful drug treatment in these models could be used as

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 DEVELOPMENT OF THERAPEUTICS candidate biomarkers indicating poxvirus control and further evaluated in nonhuman primates infected with variola. Similarly, the pharmacokinetic and pharmacodynamic properties of the drug in these models need to reflect those in humans. If such biological parameters can be validated, it may eventually be possible to use these measures in developing an alternative to testing with live variola virus. For example, a VIG formulation containing monoclonal antibodies to variola B6 could be tested in mice infected with a vaccinia virus expressing the orthologous vaccinia B5. An ectromelia infection of mice could perhaps have a profile similar to a variola infection of mice, resulting in the same alterations in host response whether the challenge virus was ectromelia or variola. Thus, host responses to drug treatments after ectromelia infection could serve as surrogate biomarkers for efficacy in the absence of live variola infection. Nevertheless, in accordance with Table 4-1 in Chapter 4, biomarkers developed in nonhuman primate models would be more likely to reflect the disease progression in human beings and therefore make better surrogates for disease progression in antiviral testing studies. Any predictions about drug activity against variola would have to be made with great caution. REFERENCES Aldaz-Carroll, L., J. C. Whitbeck, M. Ponce de Leon, H. Lou, L. Hirao, S. N. Isaacs, B. Moss, R. J. Eisenberg, and G. H. Cohen. 2005. Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R. Journal of Virology 79(10):6260–6271. Aldaz-Carroll, L., Y. Xiao, J. C. Whitbeck, M. Ponce de Leon, H. Lou, M. Kim, J. Yu, E. L. Reinherz, S. N. Isaacs, R. J. Eisenberg, and G. H. Cohen. 2007. Major neutralizing sites on vaccinia virus glycoprotein B5 are exposed differently on variola virus ortholog B6. Journal of Virology 81(15):8131–8139. Arumugham, B., H. J. Kim, M. N. Prichard, E. R. Kern, and C. K. Chu. 2006. Synthesis and antiviral activity of 7-deazaneplanocin A against orthopoxviruses (vaccinia and cowpox virus). Bioorganic & Medicinal Chemistry Letters 16:285–287. Bailey, T. R., S. R. Rippin, E. Opsitnick, C. J. Burns, D. C. Pevear, M. S. Collett, G. Rhodes, S. Tohan, J. W. Huggins, R. O. Baker, E. R. Kern, K. A. Keith, D. Dai, G. Yang, D. Hruby, and R. Jordan. 2007. N-(3, 3a, 4, 4a, 5, 5a, 6, 6a-Octahydro-1,3-dioxo-4,6- ethenocytoprop[f]isoindol-2-(1H)-yl)carboxamides: Identification of novel orthopoxvirus egress inhibitors. Journal of Medicinal Chemistry 50:1442–1444. Baker, R.O., M. Bray, and J. W. Huggins. 2003. Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections. Antiviral Research 57(1-2):13–23. Bartee, E., M. R. Mohamed, M. C. Lopez, H. V. Baker, and G. McFadden. 2009. The addition of tumor necrosis factor plus beta interferon induces a novel synergistic antiviral state against poxviruses in primary human fibroblasts. Journal of Virology 83(2):498–511. Becker, M. N., M. Obraztsova, E. R. Kern, D. C. Quenelle, K. A. Keith, M. N. Prichard, M. Luo, and R. W. Moyer. 2008. Isolation and characterization of cidofovir resistant vaccinia viruses. Virology Journal 5:58.

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