Nucleic Acid-based Drugs against Emerging Zoonotic Viruses

Transcription

1 Nucleic Acid-based Drugs against Emerging Zoonotic Viruses Jonathan P. Wong * Defence R&D Canada, Suffield Research Centre, Ralston, Alberta, Canada *Corresponding author. Tel: Fax: jonathan.wong@drdcrddc.gc.ca Abstract Global outbreaks of diseases by zoonotic viruses have steadily increased in recent years. These emerging viruses are hard to protect against due to lack of approved antiviral drugs and vaccines against them. The aim of this review is to discuss how advances in genomics, rational drug design and innate immune signaling can contribute to the design of nucleic acid-based drugs which can play a significant role in combating these emerging threats. Specifically, sirnas, antisense oligonucleotides and micrornas have specific antiviral activity mediated by gene-silencing, and toll-like receptor agonists eliciting broad-spectrum innate and antiviral immune responses. This review will discuss the state of development, safety and efficacy of preclinical and/or clinical studies, and outline next steps necessary to full licensure. Defence Research and Development Canada External Literature DRDC-RDDC-2015-P051 August 2015

2 Introduction New viruses with pandemic potential, particularly from zoonotic sources, continue to emerge globally with increasing frequency (1). As evident from the current ebola outbreaks in West Africa, these viruses can emerge without warning, kill thousands of people with a high case fatality rate, cross international borders with ease, cause massive public panic, and result in economic and political instability. Among all the zoonotic viruses infecting humans, none is more deadly than pandemic influenza. Since its first isolation and identification, influenza had caused 6 pandemics, including the Spanish flu which was believed to have killed million people worldwide (2). In the past 10 years, new variants of influenza viruses which emerged from zoonotic sources which are pandemic or have pandemic potential include the highly pathogenic H5N1 avian influenza, the swine pandemic H1N1 influenza, and the H7N9 influenza virus. Licensed antiviral drugs and vaccines represent the most effective first line defence to combat flu pandemics. However, vaccines are most likely to be unavailable against new variants of pandemic influenza, and will require significant time to be developed, tested and produced. Antiviral drugs include adamantanes (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) are widely licensed and available for use in an influenza pandemic, but uncertainty about which influenza strain(s) would cause pandemics, the degree of viral drug susceptibility and antiviral drug resistance could significantly adversely impact the suitability and usefulness of licensed drugs in flu pandemics. In view of this uncertainty, there is compelling case to argue for the possible role of experimental drugs to be used to combat future flu pandemics. When used appropriately alone, or in combination with licensed drugs, these experimental drugs can potentially have a significant role to play in improving treatment options & pandemic preparedness against, and in reducing mortality & morbidity from, pandemic influenza. There are currently many experimental drugs which are in various stages of preclinical and clinical development against influenza. Since it is not feasible to cover all of these, the scope of this review is to highlight a very selective number of drugs which are promising based on safety and efficacy from animal or human studies, and discuss them in the context of their mechanisms of action, preclinical or clinical efficacy and current state of development. These drugs are designed for treating or preventing seasonal influenza, but their suitability for use against pandemic influenza will also be addressed, particularly in cases where preclinical efficacy against pandemic influenza viruses are known. From lessons learned from the current ebola crisis, this review will also bring to the attention for the paramount importance of proactive efforts to accelerate the advanced development for these experimental drugs to full licensure, whether through the conventional drug development path or through fast track development under the pandemic influenza guidelines. For the purpose of this review, experimental drugs against influenza may be divided generally into 2 main groups: virus- or host-specific. Virus-specific are antiviral drugs which specifically target virus

3 proteins, functions or viral replication. Host-specific antiviral drugs generally target the one or more components of the host s innate immunity or cellular functions, but often confer a broader antiviral response. Virus-specific antiviral drugs Virus-specific drugs are drugs which are designed to target specific virus proteins, structures and viral replication. All existing widely licensed anti-influenza drugs including amantandanes (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) are virus-specific. Neuraminidase inhibitors (peramivir) Peramivir (RWJ ), a sialic acid analogue with a cyclopentane structure, is an experimental antiviral drug that has shown potent antiviral activity against both influenza A and B viruses (3). Perimivir is an anti-influenza drug and a potent inhibitor of influenza viral neuraminidase (NA). This novel NA inhibitor works by interfering with the release of progeny influenza virions from the surface of infected host cells. By doing so, peramivir and other NA inhibitors prevent virus infection of new host cells and thereby curtail the spread of infection in the respiratory tract. Peramivir binds to NA very tightly and causes a prolonged inhibiting of NA activity, allowing a lower frequency of dosing, and thus lessen the likelihood of virus developing drug resistance. When the NA inhibiting activity of peramivir was measured and compared to oseltamivir and zanamivir (licensed NA inhibitors) in a tissue culture assays against a number of clinically significant isolates of influenza A and B viruses, peramivir was found be more potent against H1N1 and H5N1 isolates, and had similar antiviral activity against H3N2 and B viruses (4). When peramivir was evaluated for its antiviral efficacy against multiple influenza A and B strains using a lethal murine influenza virus infection model, it was found that peramivir was more effective than oseltamivir for the 3 out of the 4 strains tested, and was equally effective against the fourth strain (5). Treatment with peramivir (100 mg/kg/dose) in these mice provided complete protection (100% survival rate) against all 4 strains (A/Shangdong/09/93 [H3N2], A/Victoria/3/75 [H3N2], A/NSW/22 [H1N1] and B/HongKong/05/72) (5). The antiviral efficacy of peramivir was also evaluated in mice against avian influenza A H5N1 and H9N2 viruses. Treatment of mice with 10 mg/kg/dose provided complete protection against influenza A/HK/156/97 (H5N1) while complete protection was achieved with 1 mg/kg/dose against mouse-adapted A/quail/HK/G1/97 (H9N2) (6). At these doses, peramivir was well tolerated in these animals without any sign of toxicity. Based on the efficacy and tolerability of peramivir obtained from the preclinical studies, clinical trials of peramivir were initiated. A number of phase 1 and 2 studies on peramivir were conducted which demonstrated safety and dose tolerability in normal healthy subjects, and as well as safety, dose tolerability and antiviral efficacy in human subjects with uncomplicated seasonal influenza infection. In one such study, the safety and efficacy of one intravenous dose of peramivir was evaluated in outpatients with uncomplicated seasonal influenza virus

4 infection. Treatment with 300 mg or 600 mg peramivir was well tolerated with no serious adverse events, and significantly reduced the time to alleviation of influenza symptoms at both 300 mg and 600 mg dose compared to the placebo (7). On November 2009, the US FDA issued an Emergency Use Authorization (EUA) for the use of intravenous peramivir as part of the emergency public health response to 2009 pandemic influenza A (H1N1) pdm09 virus [ph1n1]. Under this EUA, peramivir was authorized for adult and ph1n1 pediatric patients who was not responding to antiviral therapy and could not suitable to receive oral or inhaled NAI. In one such study conducted for patients hospitalized with severe ph1n1 viral pneumonia under EUA in the US, intravenous peramivir was found to be well tolerated, hospitalized adults and children treated with the drug for 1-14 days (median duration 10 days), the 14-, 28- and 56-day survival rates were 76.7%, 66.7% and 59% (8). The results from the other clinical studies assessing the safety and efficacy of peramivir for the treatment of ph1n1 virus were mixed. In one such trial conducted during the 2009 ph1n1 pandemic in Japan, intravenous peramivir (600 mg once daily) was given to children <16 years old to assess the safety and efficacy of the drug in pediatric infected patients (9). In this study, 62.6% of the patients developed adverse events which were mild or moderate, with 14% of patients developing severe adverse effects which include decrease in neutrophil count. In terms of efficacy, peramivir treatment resulted in decreasing the proportion of virus positive patients from 100% at the start of the study to 78.2% at day 2 post treatment to 7.1% at day 6 post drug treatment (9). In another trial involving 57 critically ill ph1n1 patients in ICU, the use of intravenous peramivir was associated with significantly higher mortality rate and higher acute respiratory distress syndrome, pneumonia and requirement for mechanical ventilation, compared to other critically ill patients who did not receive peramivir (10). Peramivir is an approved antiviral drug in Japan and South Korea. On December 22, 2014, US FDA announced the approval of intravenous peramivir for the treatment of influenza patients who have trouble taking oral or inhaled antiviral. Another neuraminidase inhibitor against influenza which was approved in Japan is laninamivir octanoate (CS-8958). Laninamivir octanoate is normally given to patients as a single inhalation. In a randomized clinical trial in Japan, it was found to reduce the median time for symptom alleviation compared to oseltamivir in patients infected with oseltamivir-resistant strain. Its efficacy against H3N2 strains is yet to be confirmed. It has yet to receive regulatory approval in the US. RNA polymerase inhibitors (favipiravir) Favipiravir (T-705, 6-fluro-3-hydroxy-2-pyrazinecarboxamide) is a drug that exerts its antiviral activity by selectively inhibit the RNA-dependent RNA polymerase (RdRP) of influenza and of many other RNA

5 viruses (11, 12). Developed by Toyama Chemical Co, Ltd. (a subsidiary of Fujifilm Holding Corp., Japan), favipiravir inhibition of influenza replication is mediated by the cellular enzymatic conversion of favipiravir (prodrug) into its active phosphoribosylated form favipiravir-ribofuranosyl-5 -triphosphate (favipiravir-rtp). Cellular mechanistic studies suggested that favipiravir-rtp is then misincorporated into nascent viral RNA, thereby inhibiting RNA strand extension (13). Additionally, favipiravir-rtp can also directly inhibit influenza viral RdRP thereby suppressing viral RNA replication and transcription (14) Favipiravir is a promising potent anti-influenza drug because it has been shown to inhibit all serotypes and strains of influenza A, B and C viruses tested so far, as well as demonstrating antiviral activity against influenza strains which are resistant to currently licensed neuraminidase inhibitors (15). The therapeutic efficacy of favipiravir is well established against many strains of influenza A in both in vitro cell culture as well as in vivo mouse efficacy studies (15, 16). In mouse studies, favipiravir orally administered 2 or 4 times for 5 days (100 mg/kg/day) provided complete therapeutic protection to mice against 100 lethal doses of influenza A/Victoria/3/75 (H3N2), A/Duck/MN/1525/81 (H5N1), and 90% protection against multiple lethal doses of A/Osaka/5/70 (H3N2). Due to its unique mechanism of action, favipiravir has been shown to demonstrate broad-spectrum activity in experimental animal models against many other RNA viruses, including ebola, western equine encephalitis, Rift Valley fever, yellow fever, respiratory syncytial viruses, among many others (15) The safety and drug tolerability of favipiravir has been extensively evaluated in over 1400 human subjects tested in many countries in the world (15 ). A phase 3 study was completed in Japan and two phase 2 studies have been completed in the US. Patients are currently being recruited for a phase 3 study in the US, according to the website information found on A double-blinded, randomized phase 3 clinical trial for favipiravir is currently being conducted in January, 2015 for the treatment of uncomplicated seasonal influenza in adults. The results of this phase 3 trial are expected to be available when this trial is completed in February 2015 The advanced state for development for favipiravir, supported by safety and efficacy data from preclinical and clinical studies, would place favipiravir to be closest among the various experimental drugs to full licensure in the US and EUA. Favipiravir will be particularly useful if and when used in flu pandemics caused by influenza strains which are resistant to the neuraminidase inhibitors, as preclinical efficacy studies had demonstrated potent activity of favipiravir against these resistant strains (15, 17). Furthermore, its broad-spectrum antiviral activity will be beneficial to combat new variants of influenza viruses, regardless of pandemic or seasonal strains. In March 2014, favipiravir won approval from the Japanese regulators for stockpiling for possible use against future influenza pandemic. Thiazolides (Nitazoxanide) Nitazoxanide was originally developed as an anti-protozoal agent, but had been repurposed for the treatment of influenza. A randomized, double-blinded, phase 2b/3 clinical trial in the US showed that patients who received 600 mg nitazoxanide orally showed reduced duration of clinical symptoms and viral shedding compared to placebo control group. Its anti-influenza activity is thought to be mediated

6 by selectively blocking the maturation of the viral hemagglutinin protein at a post translational level, thus interfering with virus assembly and release. Therapeutic antibodies With highly fatal viral infections where there are no effective licensed antiviral drugs, neutralizing antibodies can provide rapid therapeutic benefits to patients. Two ebola-infected US health care workers attributed treatment with neutralizing antibodies (ZMapp) for their recovery. Experimental therapeutic antibodies suitable for use in influenza pandemics must be non-immunogenic, can be produced in large quantities within a short time window, and can protect against multiple strains, including drug resistant strains. Advances in antibody engineering have paved the way for the design of antibody-based therapeutics which can meet these requirements. For example, monoclonal antibodies can be generated from transgenic mice engineered to produce fully humanized antibodies following immunization with a consensus-sequence viral antigen. In one such study, several humanized monoclonal antibodies were generated following immunization of transgenic mice using a consensussequence of the influenza M2 protein (18). Among which is one monoclonal antibody (Z3G1) with high affinity binding to the majority of M2 protein from natural viral isolates, including the highly pathogenic avian strains (19). Passive immunotherapy with this monoclonal antibody protected mice from a lethal virus challenge and resulted in significant reduction in viral replication, when the antibody was administered to the mice prophylactically or therapeutically. The treatment with Z3G1 can be administered at late as 3 days post infection, and it still offered significant protection of mice against the lethal virus challenge. Furthermore, Z3G1 offered protection in mice infected with different strains of influenza virus, including amantadine- and oseltamivir-resistant strains (19). As well, Z3G1 reduced lung pathology in monkeys infected with a 2009 ph1n1 strain (19). Another humanized monoclonal antibody with neutralizing activity against the ph1n1 influenza virus was expressed in a mammalian cell line (20). Another promising therapeutic antibody is an adenovirus vector-expressed neutralizing monoclonal antibody to influenza A virus, which can be delivered to the respiratory tract or the site of infection. When delivered intranasally, this adenovirus expressed antibody provided complete protection to mice against 100 lethal doses 50 of 3 clinical isolates of the H5N1 and two clinical isolates of H1N1, and resulted in significant virus load reductions (21). Similar level of protection was achieved in ferrets challenged with lethal doses of ph1n1 and H5N1 (21). These preclinical data suggest that it is possible to design antibody-based therapeutics which are safe (at least in experimental animals), efficacious and protect against multiple strains, and they do offer the potential to prevent and treat influenza virus infection by pandemic strains. In addition, it is also possible to isolate neutralizing antibodies from

7 sera of recovering influenza patients, and these will likely have therapeutic effectiveness which will protect others during pandemics. There remain many challenges from the use of neutralizing antibodies for the prevention and treatment of pandemic influenza in humans. The scale up production of these antibodies in mammalian cell systems or expressed in plants can be problematic. This problem is highly evident in the production of ZMapp antibodies expressed in tobacco plants during the current ebola crisis. Secondly, the safety and functionality of these exogenously produced antibodies will have to tested and confirmed in clinical studies prior to use. With monoclonal antibodies, cocktail combinations of two or more antibodies against various viral protein sites may be required for optimum protection. Finally, matching the neutralizing antibodies against the prevailing or prevalent pandemic influenza strains will be required. Host-specific drugs Host-specific drugs are drugs which exert their antiviral effects by targeting one or more components of the host s innate immune or antiviral responses, rather than targeting the virus directly. Development of host-specific drugs may represent a much-needed paradigm shift in anti-influenza drug development as all licensed drugs are virus-specific. In general, host-specific drugs exert a broader biological (immunological or antiviral) effect in the body than virus-specific drugs, and are considered to be less likely to give rise to drug resistance. Fludase (DAS181) Fludase (DAS181) is a novel anti-influenza virus agent and a recombinant fusion protein with sialidase activity (22). It works by catalyzing the removal of sialic acid-containing receptor from epithelial cells from the respiratory tract, thus preventing and inhibiting the attachment and replication of influenza virus in the respiratory tract. Fludase has been extensively evaluated in tissue culture and animal studies, and it has shown excellent therapeutic efficacy against some of the highly pathogenic avian influenza viruses, including the HPIA (22), H7N9 (23) and the pandemic ph1n1 virus (24). In one study in mice infected with the highly virulent influenza H7N9 virus, once daily dosing of fludase by intranasal administration initiated early after otherwise lethal infection provided complete protection to animals (23). Fludase treatment also protects them from exhibiting severe infection symptoms, including body weight loss. Delayed fludase treatment at 24 and 48 hours post infection resulted in % protection (23). Additionally, fludase treatment also protected mice against lethal challenge with oseltamivir-resistant strain of influenza A virus (25).

8 Fludase has completed 3 phase 1 trials in normal healthy volunteers (clinical.trials.gov/show/nct ), and a phase 2 clinical trial in laboratory confirmed influenza patients (26). Overall, fludase was found to be well tolerated in normal healthy volunteers in phase I trial, and in laboratory-confirmed influenza patients (26). The incidences of severe adverse events (SAE) were found low in these trials, with the most common laboratory abnormality was the transient elevation of alkaline phosphatase observed in both trials (26). Furthermore, 2 patients in the phase 2 study experienced SAE in both the placebo and the single-dose group, none of the patients in the multiple-doses had SAE. In terms of antiviral efficacy, fludase was assessed for efficacy in patients infected with seasonal influenza H3N2, ph1n1 and influenza B (26). Statistically significant antiviral efficacy of fludase was observed in the multiple-dose group compared to the placebo group as measured by multiple virological analyses. Patients in this group showed significant reduced virus load from baseline compared to the placebo group over a 5 day period. In addition, there was a significant decrease in the time required to sustain decreased virus shedding in the multiple-dose treatment group compared to the placebo group. More efficacy studies are needed to assess the impacts of higher and more frequent dosing on clinical outcome, and on virus load and shedding. Toll-like receptor agonists (Poly ICLC and Ampligen) During influenza viral replication cycle, viral RNAs accumulate intracellularly in infected host cells. The presence of these viral RNAs are recognized and detected by toll-like receptors (TLRs) which are transmembrane signaling proteins expressed by cells of the host innate immune system. Pathogenderived genetic materials are signatory molecules detected by nucleic acid-recognizing TLRs, and these include TLR-3 (recognizes double-stranded [ds]rna), TLR-7 (single-stranded [ss]rna), TLR-8 (ssrna) and TLR-9 (unmethylated CpG motifs). The binding of viral RNAs to TLR-3, -7 and -8 triggers these TLR signaling pathways and it is a critical mechanism which mediates the host innate immune and inflammatory systems to respond and to fight the viral infections (27). Due to the pivotal role of TLRs in augmenting the host s antiviral defence, TLR agonists have become a hot area in antiviral drug designs in recent years. The potential clinical use of TLR-3 agonists as immunotherapeutic agents in infectious diseases and oncology is currently being explored (28-30). TLR-3 agonists have been shown to elicit a broadspectrum antiviral immune response against a variety of viruses (28-33). Among these are dsrnas including Poly ICLC and Ampligen. Poly ICLC is a synthetic ds RNA consisting of complementary strands of polyriboinosinic polyribocytidylic acid (IC) condensed with poly-l-lysine and carboxymethylcellulose (LC). Poly ICLC is a well-established TLR-3 agonist and when it binds to TLR-3, it induces the production of type I and II interferons, activates natural killer cells, macrophages and dendritic cells, and stimulates key antiviral enzymes such as interferon-inducible protein kinase and R 2-5 oligoadenylate synthetase (29). In a number of preclinical studies, Poly ICLC has been shown to effectively protect experimental animals against some of the deadliest viruses known, including ebola virus, Rift Valley fever virus and SARS-corona virus. Another TLR-3 agonist composed of a modified IC is Ampligen (polyi:polyc 12 U) (30, 31). Ampligen has been tested in phase 3 clinical studies in the US for chronic fatigue syndrome.

9 Ampligen has shown broad-spectrum antiviral activity similar to Poly ICLC (30). ampligen is an effective adjuvant for avian influenza H5N1 vaccine (31). Against influenza, The prophylactic and therapeutic efficacy of Poly ICLC has been evaluated against different strains of influenza A viruses. In mice, pre-treatment with two intranasal doses of Poly ICLC (1 mg/kg/dose) given 48 hours apart provided the optimum prophylactic protection. Against influenza A/PR/8/34 (H1N1), pretreatment with Poly ICLC has been shown to provide 100% survival rates in mice when given prophylactically as far as 12 days prior to a multiple lethal dose virus challenge (32). Nasal spray of poly ICLC was also found to protect ferrets against influenza A/PR/8/34 (unpublished results). Pretreatment with Poly ICLC was also found to provide high level of protection in mice against the highly pathogenic avian influenza A/H5N1 virus (33). Poly ICLC can be administered intramuscularly, intranasally or intravenously to the host, with two doses (1 mg/kg body weight) given 48 hours apart providing optimum protection in animals (32). However, when administered to mice 8 and 48 hours post virus challenge, the antiviral efficacy of Poly ICLC decreased from 100% (pre-treatment) to 40%. In terms of current state of development, Poly ICLC had been shown to be safe and well tolerated in normal healthy volunteers in a nasal phase I clinical trial in the US. Phase II clinical studies to evaluate the efficacy of Poly ICLC in an appropriate influenza trial in the US are envisaged. Drugs which treat cytokine storm induced by pandemic influenza viruses Highly virulent Influenza viruses are known to cause aberrant over-responsive immune responses which are linked to uncontrolled overexpression of pro-inflammatory cytokines/chemokines (acute hypercytokinemia). This cytokine storm effects, manifested by hyperinduction of inflammatory and apoptotic cytokines, infiltration and activation of T-effector cells, can lead to lung injury, multiple organ failures and shock, and are associated with high rate of mortality in young and otherwise healthy people infected by these viruses (34). Influenza viruses which cause cytokine storm include the Spanish flu, the HPIA and the pandemic 2009 H1N1 viruses. Treatment strategy required to reduce mortality in these patients will encompass the use of an effective antiviral drug to block viral replication, but may also involve treating the cytokine storm-associated effects with an anti-inflammatory drug that does not impede the body s ability to fight the viral infection. Towards this end, a number of anti-inflammatory drugs had been evaluated for the treatment of cytokine storm in influenza infections. Glucocorticoids treatment for the suppression of cytokine did not protect mice from lethal infection with HPIA (A/Vietnam/1203/04 H5N1) (35). In case studies in humans during the H5N1 outbreaks in South East Asia, patients who were treated with steroids such as dexamethasone, methylprednisolone, with or without oseltamivir, were found to have high mortality rates, and steroid treatment did not improve clinical outcomes (36). Recently, the experimental use of sphingosine-1-phosphate-1 receptor (S1P1R) agonists for the suppression of cytokine storm and for the protection against pathogenic influenza virus infection in mice

10 has been described (37). Novel therapy with SIP1R agonists (CYM5442 and AAL-R) in mice following infection with human pandemic influenza A/Wisconsin/WSLH34939/09 inhibits the cellular and cytokine/chemokine responses to limit the immunopathogenic damage, as well as providing significant protection (82% survival, 23/28 mice) against an otherwise lethal virus challenge (38). This level of protection was higher when compared to treatment with the antiviral drug oseltamivir (50% survival, 14/28 mice), although combining AAL-R and oseltamivir provided the highest level of protection (96% survival rate, 27/28 mice) (38). Mechanistic studies in mice have demonstrated that the efficacy of S1P1R agonist in suppressing cytokine storm is achieved through the systemic inhibition downstream of the myeloid differentiation primary response gene (MyD88) and IFN-β promoter stimulator-1 signaling (39). MyD88 of the innate immune system was identified in the study as the adaptor molecule of the TLR signaling pathway responsible primarily for the cytokine amplification following pathogenic influenza virus challenge. These preclinical data suggest the potential therapeutic effects of S1P1R agonists for the use in treatment of potentially fatal cytokine storm conditions, and will therefore improve clinical outcomes and significantly reduce mortality-associated with the highly pathogenic or pandemic influenza patients. In terms of future development of SIP1R agonists, it is not known if and when they will undergo safety and dose tolerability testing in phase 1 clinical trial. If these potential drug products are shown to be safe and well tolerated in phase I trial, there is compelling justification for these drug products to be fast tracked for regulatory approval. Having these drugs licensed will greatly enhance our pandemic preparedness not only against influenza, but may have potential applicability to combat other emerging viral diseases which induce cytokine storm, including ebola, MER-COV and SARS. Conclusions Many public health and infectious disease experts acknowledge that the world is ill-prepared for the next deadly influenza pandemics. The unpredictability of pandemic influenza, the global rise in drug resistance, and constant virus mutations represent the most serious challenges to influenza pandemic preparedness strategies. Despite this pressing unmet need, there has not been a new anti-influenza drug approved for worldwide use since There are many regulatory hurdles to clinical development of new antiviral drugs, and development of new anti-influenza is an expensive and complex process where failure rate is high. Until new and more effective antiviral drugs are licensed, experimental drugs will continue to be valuable as prophylactic and therapeutic agents should deadly pandemic influenza which is drug resistant were to emerge. Pandemic influenza is a very complex disease which involves interplays between virus-target cells interactions, innate immune responses and viral genetics. For example, licensed drugs may be effective inhibiting viral replication but may not treat respiratory failure of patients due to cytokine storm. Therefore, management and treatment of pandemic influenza often may require a multi-prong approach, thus opening the door for innovative treatment options. Furthermore, the best weapons to combat emerging pandemic influenza viruses may come from combination therapy of existing drugs or some combinations of licensed and experimental drugs.

11 This review highlighted a few examples of selected promising experimental drugs currently in development. They represent treatment options which can be added to the existing arsenal of antiviral drugs needed to combat deadly or pandemic influenza. These virus-specific and host-specific experimental drugs and their modes of action are summarized and diagrammatically depicted in Figures 1a and 1b, respectively. As shown, these experimental drugs primarily have mechanism of actions which are different from existing ones (with the exception of neuraminidase inhibitor peramivir), and are therefore more likely to be effective against influenza viruses which are resistant to amantadine and oseltamivir. Of particular significance are novel experimental drugs which are host specific and target the host s innate immune responses and suppress the cytokine storm. They represent a new frontier in antiviral development against severe influenza, and potentially play an important role in reducing cytokine storm-associated mortality especially in young and otherwise healthy people infected with pandemic influenza. More preclinical and clinical studies will need to be conducted to assess the safety and efficacy of these experimental drugs before they can be considered for clinical, and/or possibly for emergency use, during influenza pandemics. Proactive efforts are needed now to advance them for regulatory review for full licensure, or policies and guidelines for emergency use should be in place before the next influenza pandemic emerges. Future Perspective The world s ability to combat future pandemic influenza has significantly improved with valuable lessons learned from numerous emerging virus outbreaks, and the steady pace of development for novel antiviral drugs against influenza viruses (both seasonal and potential influenza). Looking forward, it is envisaged there will be a tendency to invest more research and development efforts into advancing antiviral drugs with more robust mechanism of action which will less likely to give rise to drug resistance. This paradigm shift is needed to minimize the perennial problem of influenza virus developing drug resistance. To achieve this, innovative approaches will need to be developed towards host-specific rather than virus-specific drugs, and novel treatment regimens towards addressing virusinduced hypercytokinemia and bacterial pneumonia. Executive Summary Unpredictability of pandemic influenza, increasing drug resistance to anti-influenza drugs and lack of medical countermeasures to treat cytokine storm are compelling reasons to consider the use of safe and efficacious experimental drugs for use in future influenza pandemics These drugs may likely enhance our influenza preparedness against future pandemics, and potentially reduce the impact and mortality from influenza infections and associated complications

12 There are a number of promising experimental drugs which are currently in various stages of preclinical and clinical development, and among these are leading antiviral drugs which have characteristics which render them potentially useful during influenza pandemics Experimental drugs against influenza can be generally divided into 2 groups according to their drug targeted actions: Virus or host-specific Experimental drugs which target the influenza viruses include novel neuraminidase inhibitor peramivir, RNA-dependent RNA polymerase inhibitor, favipiravir, and therapeutic antibodies which target the conserved virus sequences Host specific experimental drugs include toll-like receptor-3 agonists including Poly ICLC and ampligen, and they have the potential to elicit broad-spectrum innate immune responses which can protect against drug-resistant and/or mutated pathogenic influenza virus strains Cytokine storm is associated as a primary cause of shock, respiratory failure and mortality in patients infected with highly pathogenic or pandemic influenza. S1P1R agonist has been shown in animal studies to be effective in suppressing cytokine storm and reducing mortality without affecting the host s ability to control virus clearance Experimental drugs which have well documented safety and efficacy profiles but have not yet been fully licensed may play an important role in complementing existing drug to combat emerging or pandemic causing influenza viruses Proactive efforts to accelerate the advanced development of selected leading experimental drugs to full licensure are needed now References 1. Smith, KF, Goldberg M, Rosenthal S et al. Global rise in human infectious diseases. J. Royal Soc. Interface 11(101): doi: /rsif (2014) 2. Johnson NP, Mueller J. Updating the accounts: global mortality of the Spanish influenza pandemic. Bull. Hist. Med. 76: (2002) 3. Young D, Fowler C, Bush K. RWJ (BCX-1812): A novel neuraminidase inhibitor for influenza. Phil. Trans. R. Soc. Lond. B. 356: (2001).

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16 32. Wong JP, Saravolac EG, sabuda D et al. Prophylactic and therapeutic efficacies of poly(iclc) against respiratory influenza virus infection in mice. Antimicrob. Agents Chemother. 39: (1995) 33. Wong JP, Christopher ME, Salazar AM et al. Nucleic acid-based antiviral drugs against seasonal and avian influenza viruses. Vaccine 25: (2007) 34. Osterholm, MT. Preparing for the next pandemic. New Engl. J. Med. 352: (2005) 35. Salomon R, Hoffmann E, Webster RG. Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection. Proc. Nat. Acad. Sci. USA 104(30): (2007) 36. Carter MJ. A rationale for using steroids in the treatment of severe cases of H5N1 avian influenza. J. Med. Microbiol. 56: (2007) 37. Oldstone MB, Rosen, H. Cytokine storm plays a direct role in the morbidity and mortality from influenza virus infection and is chemically treatable with s single sphingosine-1-phosphate agonist molecule. Curr. Top. Microbiol. Immunol. 378: (2014). 38. Walsh KB, Teijaro JR, Wilker PR et al. Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus. Proc. Natl. Acad. Sci. USA108 (29): (2011). 39. Teijaro JR, Walsh KB, Rice S et al. Mapping the innate cascade essential for cytokine storm during influenza virus infection. Proc. Natl. Acad. Sci. USA 111(10) (2014). Key words Pandemic influenza, experimental drugs, prevention and treatment, safety, efficacy, cytokine storm, drug resistance and mechanism of action.

17 Figure Legends 1. A simplified diagrammatic depiction of selected experimental drugs discussed in this review and their modes of antiviral action for (a) virus-specific and (b) host-specific experimental drugs. For detailed description of their antiviral activity and modes of action, please refer to the main text of the review. Acknowledgments The author wishes to acknowledge Dr. Les Nagata for his review of this manuscript, and Shelley Ewing for her wonderful graphic illustrative work. Financial and competing interest disclosure The author s research is supported financially by the Department of National Defence under the Government of Canada. The author has no other relevant affiliation or financial involvement with any organization or entity with financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

18 Figure 1A.

19 Figure 1B.