Influenza antivirals currently in late‐phase clinical trial

Influenza antiviral drugs are important for the control of influenza, most specifically for the treatment of influenza patients with severe disease following infection with a seasonal influenza virus, a newly emerging influenza strain, or in the event of a pandemic. Many influenza antivirals that are currently under investigation in late‐stage clinical trials differ in their mechanism of action compared to drugs currently licensed for the treatment of influenza. Nitazoxanide and DAS181 target components of the host cell and alter the ability of the virus to replicate efficiently, while small molecule drugs such as T705, JNJ63623872 and S‐033188 bind to the viral polymerase complex and restrict viral replication. Monoclonal antibodies that are currently in clinical trial for the treatment of influenza most commonly are targeted to the stem region of the haemagglutinin molecule. Early findings from animal models and in vitro studies suggest that many of the new antiviral drugs when tested in combination with oseltamivir have improved effectiveness over monotherapy. Clinical trials assessing both monotherapy and combination therapy are currently under investigation. It is hoped that as new antivirals are licensed, they will improve the standard of care and outcomes for influenza patients with severe disease.


| INTRODUCTION
Although influenza vaccines remain the most effective means to prevent seasonal influenza, they typically provide suboptimal protection in high risk groups. 1 In addition, vaccines may be completely ineffective in the event of an antigenic mismatch between viruses in the seasonal vaccine and those circulating in the community. 2,3 In comparison, influenza antiviral drugs typically remain effective against antigenic drift variants or even newly emerged pandemic viruses as they target highly stable or conserved parts of the virus. In many countries, antiviral medication is administered to hospitalised patients with severe influenza illness and is stockpiled as part of pandemic preparedness plans. [4][5][6] Some countries, including Japan and the USA, also use substantial amounts of influenza antivirals for the treatment of uncomplicated influenza infection in otherwise healthy individuals. 7,8 Compounds from only two classes of influenza antivirals are licensed for use in many countries. These are the adamantanes and neuraminidase inhibitors (NAIs). The adamantane compounds, amantadine and rimantadine, target the viral M2 protein required for virus uncoating during replication. 9 However, these compounds are no longer recommended for treatment of seasonal influenza A viruses due to widespread viral resistance. 10 NAIs impair the release of virus from infected host cells, and although the frequency of NAI resistance in currently circulating strains is low (<1%), 11 resistance to oseltamivir, the most widely used NAI, was widespread amongst former seasonal H1N1 viruses in 2008. 12,13 More recently, localised clusters of oseltamivirresistant H1N1pdm09 viruses have been detected. 14,15 Aside from concerns regarding resistance, the effectiveness of the NAIs is limited when delivered >48 hours after the onset of symptoms. Given these factors, there remains an important need for the development of influenza antiviral drugs that improve treatment effectiveness and can be delivered easily to patients with severe illness, remain effective when delivered late in the progression of disease and that have a low propensity for selecting for viral resistance.
This review summarises the new influenza antiviral drugs currently in phase II or III clinical development (as described on www.clinicaltrials. gov). This includes two compounds (nitazoxanide (NTZ) and DAS181 (Fludase)) that target features of the host cell that are critical for influenza replication, three compounds (T705 (favipiravir), JNJ63623872 (VX-787) and S-033188) that target components of the viral polymerase complex and a number of monoclonal antibodies that are being investigated as therapeutics in the treatment of influenza virus infection. A list of these antivirals including their mechanism of action and clinical trial status is provided in Table 1. Adjunctive therapies such as macrolides, nonsteroidal anti-inflammatory drugs and mTOR inhibitors that have been investigated in combination with antiviral therapy are not discussed in this review.

| Nitazoxanide
NTZ is a compound of the thiazolide class of broad-spectrum antiviral drugs that is orally administered and rapidly deacetylated in the blood to the active metabolic form tizoxanide (TIZ). 16 NTZ was originally developed and licensed as an antiprotozoal/helminth drug and remains the only licensed treatment for Cryptosporidium infections. 17 In addition, NTZ/TIZ is also effective against a range of bacterial 18 and viral infections. 19 NTZ/TIZ was first identified to have the potential for antiviral properties during clinical trials for Cryptosporidium treatment in patients with AIDS. 20 Since that time, NTZ/TIZ has been shown to inhibit various viruses including gastrointestinal viruses like rotavirus, and influenza viruses, hepatitis B and C viruses. 19 The mode of action of NTZ/TIZ against these viruses appears to differ. For rotavirus, TIZ interferes with the VP7 structural protein formation, while for hepatitis C, TIZ aids the activation of protein kinase R (PKR) resulting in downstream activation of the innate immune pathway. 19 For influenza, TIZ exerts its antiviral activity by interfering with the assembly of the viral haemagglutinin (HA). 21 TIZ inhibits influenza virus replication by impairing the trafficking of the HA from the endoplasmic reticulum (ER) to the Golgi, preventing the exit of mature virus particles from the host cell. 22 TIZ also inhibits HA maturation by blocking HA terminal glycosylation which occurs prior to endoglycosidase H digestion of the HA terminal. 16 Second generation thialozide compounds that are structurally related to NTZ have also been shown to have broad-spectrum antiviral activity. Some of these derivatives demonstrate improved bio-availability compared to NTZ and were shown to stimulate innate immune responses to reduce HIV replication in vitro. 23 One of the thialozide compounds, the amino ester prodrug derivative RM5061, is now undergoing Phase I clinical trials. 24 In vitro studies have identified that NTZ/TIZ has antiviral activity against a range of different influenza A (H1, H3, H5, H7) and B viruses. 22,25,26 In addition, a combination of oseltamivir and NTZ/ TIZ in vitro has a synergistic effect against both human and avian influenza A viruses, including an oseltamivir-resistant virus. 27 The potential benefit in using a virus-targeting antiviral (oseltamivir) in combination with the host-targeting antiviral (NTZ) is the improvement in the effectiveness of influenza treatment as well as the reduction in the likelihood of selecting for resistance. There is currently no published data available on the effectiveness of NTZ treatment of influenza in animal models. Results from a phase IIb/III human clinical trial demonstrated that 600 mg NTZ twice daily (started within 48 hours of symptom onset) reduced symptom duration by 36 hours.
Furthermore, this dosage regime also reduced the infectious viral load by up to one log 10 compared to a placebo control. 28

| DAS181 (Fludase)
DAS181 (also known as Fludase) is a host-targeted recombinant sialidase fusion protein. It is designed to remove sialic acid receptors from glycan structures on the respiratory epithelium, thereby restricting the ability of influenza viruses to bind and enter the host cell. DAS181 is comprised of two main components, a heparin binding sequence, which anchors to respiratory epithelium, and a sialidase catalytic domain derived from Actinomyoces viscous. The active domain of the sialidase (which cleaves both α(2,6)-linked and α(2,3)-linked sialic acid receptors recognised by both human and avian/equine influenza viruses) is derived from a bacterial species that is also part of the human flora; therefore, administration is unlikely to produce adverse immunological reactions. 30 The heparin binding sequence is derived from human amphiregulin; this component binds negatively charged glycosaminoglycans and improves the binding potency to the respiratory epithelium. DAS181 is administered as an inhaled dry powder with microparticles of 5-10 μm in size, enabling the drug to access the upper and central respiratory tract, but not the lower respiratory tract. An early concern regarding the removal of sialic acid from the respiratory epithelium was that it may increase the colonisation rate infected mice. Serial passage of influenza viruses in the presence of DAS181 selected for several mutations in the HA (G137R, S136T, S186I) and NA (W438L, L38P) which resulted in viruses with increased receptor binding, coupled with significantly reduced NA on the cell surface. 38 As a result, the replication fitness of the DAS181 selected viruses was compromised. 38 Four different formulations of DAS181, which differ in particle size, have been investigated in phase I and phase II trials. A phase II trial conducted over three influenza seasons examined the effectiveness of DAS181 F02 as a single 10 mg dose or 10 mg daily over 3 days. In mice receiving the multiple dose regimen, there was a significant decrease in viral shedding and viral load up to 5 days from the commencement of treatment. 39 The safety of 20 mg doses of DAS181 F03 and F04 delivered for up to 10 days was assessed in a phase I trial. The dose was well tolerated up to 7 days, but further dosing resulted in the onset of adverse respiratory events. 40 DAS181 has also been tested in a small cohort of adults with wellcontrolled asthma. While the drug was associated with some adverse events such as chest discomfort, the drug was deemed safe for adults with uncomplicated asthma. 41

| T705 (favipiravir)
T705 (also known as favipiravir) is a substituted pyrazine derivative which inhibits the replication of a number of RNA viruses including influenza viruses. As a purine nucleoside analogue, T705 exerts its antiviral activity as a competitive substrate inhibitor of the RNAdependent RNA polymerase (RDRP) that is essential for the replication of RNA viruses. 42 Serial passage of viruses in increasing concentrations of T705 caused an increase in guanosine to adenine nucleotide mutations in the virus compared to viruses passaged in the absence of drug. 43 The increased mutation frequency, resulting in the generation of non-viable virus, is a key antiviral mechanism of T705. 43 This has also been described as the mechanism of action for the antiviral ribavirin. 44 T705 has broad-spectrum activity, also targeting and successfully inhibiting the RDRP of other viruses in vitro, and in many cases in vivo, such as West Nile virus, poliovirus and noroviruses. 45 In addition, T705 has also been shown to have some effectiveness against Ebola virus in small animal models 46

| JNJ63623872 (formerly known as VX-787)
JNJ63623872 (formerly known as VX-787) is a compound that inhibits the PB2 subunit of influenza A viruses by utilising host pre-mRNA as a primer for viral RNA synthesis during replication, a method known as "cap snatching". 56 The drug binds to key residues in the PB2 cap

| S-033188
S-033188 is a prodrug that is metabolised to an active form (known Another mAb targeted to membrane fusion is CR8020, which binds to HA stem epitopes, is active against group 2 HA subtypes of influenza A viruses and has been shown to neutralise H3N2 and H7N7 infections in vitro and in vivo. 66 CR8020 has been investigated in two placebo-controlled phase II trials in experimentally infected individuals (NCT01938352) as well as in hospitalised patients with influenza, where it was also compared with the mAb CR6261 (NCT01992276).
However, it has been shown that CR8020 targets residues that are prone to antigenic drift and therefore if escape mutants arise, then effectiveness is likely to be reduced. 67 MEDI8852 also targets the HA stalk, but has a greater potency and a broader spectrum of activity showing effective inhibition of all 16 influenza A HA subtypes. 68 MEDI8852 was shown to be superior to oseltamivir in preventing the death of mice and ferrets infected with H5N1 when treatment was administered up to 3 days post-infection. 68 MEDI8852 was shown to be well tolerated in phase I clinical trials, 69 and patients are currently being recruited for a phase IIa study to evaluate its safety in patients with uncomplicated influenza (NCT02603952).
MHAA4549A is another HA stalk targeted mAb that is also effective at binding all influenza A subtypes. 70  Another HA mAb, VIS-410, was effective at protecting mice from A(H7N9) virus challenge and improved viral clearance and spread in the lungs when delivered prophylactically. A single dose of 10 or 50 mg/kg protected all mice from death following infection with an oseltamivirresistant strain of influenza. 74 In a phase I trial, 2-50 mg/kg doses of VIS-410 were found to be safe and well tolerated by the cohort. 75 TCN032 differs from those previously discussed as it targets an epitope at the N-terminus of the matrix 2 protein which is a conserved epitope in influenza A viruses. Binding to this target has been shown to inhibit almost all influenza virus types and protected mice from lethal challenges with either H5N1 or H1N1 influenza viruses. 76 In a phase IIa human challenge study (NCT01719874), a single dose of TCN032 was found to be well tolerated. 77

| CONCLUSION
There is currently an unmet need for better influenza antiviral drugs showing substantially improved effectiveness compared to currently available drugs when treatment is initiated >48 hours after symptom onset. In addition, the propensity for the selection of viral resistance to these new compounds appears to be lower than that seen for the NAIs or adamantanes. Finally, some of the new antivirals have activity against a number of different respiratory viruses which could mean that these broad-spectrum compounds may be prescribed for a respiratory disease without the need for a specific laboratory confirmed diagnosis.

ACKNOWLEDGEMENT
The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.