4 DISCUSSION
Given the limitations of influenza vaccines and the recent rise in the number of oseltamivir-resistant strains, there remains a need to discover and develop new anti-influenza agents. In our cell culture-based screening of compound libraries, aprotinin was identified as a strong anti-influenza candidate. It has been previously reported as an anti-influenza agent in vitro ,[23] in embryonated chicken eggs,[18,24] and in mice.[25] It is currently licensed in Russia for clinical use in aerosolized form (Aerus™), primarily against seasonal H1N1 and H3N2 influenza, but it has also been tested against H2N2 and avian-like H7N9 influenza viruses.[17,19] Aprotinin is a naturally occurring non-specific inhibitor of serine proteases, including trypsin, chymotrypsin, plasmin, and kallikrein.[26] Influenza viruses require proteolytic cleavage and structural rearrangement of hemagglutinin (HA) for successful fusion with host endosomes. The HA precursor protein, HA0, is cleaved into HA1 and HA2, which are initially linked by a short peptide sequence. Trypsin-like proteases facilitate this cleavage by targeting arginine in the linker peptide of most influenza virus strains. Aprotinin is believed to inhibit HA0 cleavage by competing for the active site of these proteases.
Since previous studies have shown that aprotinin inhibited a limited number of subtypes of IAV (mainly H1N1 and H3N2), we decided to examine its antiviral activity against a broader range of influenza virus in this study. The tested strains included avian strains of IAV, an oseltamivir-resistant strain of IAV, and a strain of IBV.
Similar to previous reports, we found that aprotinin was able to inhibit the production of seasonal H1N1 and H3N2 IAVs in MDCK cells. The effects of aprotinin were either comparable or superior to the effects of oseltamivir. We also found that aprotinin could inhibit avian IAVs belonging to the H9N2, H5N2, and H6N5 subtypes in vitro at levels similar or superior to those of oseltamivir. H9N2 currently circulates in poultry and is generally avirulent or low-pathogenic. However, occasional outbreaks in poultry farms have occurred, and sporadic human infection cases have also been reported.[27] Both the H5N2 and H6N5 viruses in this study were isolated from wildfowl in South Korea. The H6N5 isolate was found to cause considerable morbidity and mortality in mice without bearing any known pathogenicity marker.[28] Meanwhile, the H5N2 isolate, adapted to and caused lethality in mice after only a single lung-to-lung passage.[29] Evidently, these isolates have the capacity to easily cross the avian-mammalian transmission barrier and may emerge as zoonotic agents in the future. The ability of aprotinin to inhibit these avian influenza viruses suggests that aprotinin may potentially be used in human outbreaks of avian influenza viruses.
We have also shown that aprotinin is able to inhibit an oseltamivir-resistant influenza A strain (A/Bris/10/07; H3N2). Additionally, similar to earlier reports of aprotinin’s activity against the B/Lee/40 and B/HK/73 viruses,[18] aprotinin shows antiviral activity against a currently circulating strain of IBV (Yamagata-like lineage, B/Seoul/32/2011). IBVs are generally less susceptible to oseltamivir, especially in children.[30,21] Because aprotinin targets a host factor required for infection, influenza viruses are less likely to develop aprotinin resistance, especially since trypsin-like proteases, the targets of aprotinin, are required for influenza virus proliferation. Therefore, the use of aprotinin may be more beneficial in the long run than the use of drugs targeted against viral components.
Aprotinin was commonly indicated as a prophylactic agent to prevent blood loss and to reduce the need for blood transfusions in cardiac bypass surgeries. However, due to safety concerns, aprotinin had been pulled out of the market in 2007; it has since been re-licensed in Canada and Europe for the same application.[31] It was generally well-tolerated in animal models and in clinical trials, and it is given at high intravenous doses for human application, suggesting that it is safe to use at high doses.[32] In our study, at least twice-daily intravenous administrations were needed for aprotinin to be protective against influenza virus infection in a mouse model. Aprotinin has a relatively short plasma half-life (0.7–2 h), and 90% of the administered dose is absorbed by the kidney in a few hours,[33] which requires aprotinin to be applied in high-dose intravenous administrations in surgeries.[32] This probably explains why once-daily intravenous administrations were not sufficient to exert inhibitory effects against the influenza virus. As such, high plasma concentrations of aprotinin may also be required to inhibit influenza viruses. However, as in the case of the licensed aerosolized aprotinin in Russia, multiple doses of intranasally administered aprotinin may be more beneficial for application against influenza virus infection in humans.[25,34,17,19] This way, aprotinin does not have to circulate systemically and will be targeted in the upper respiratory tract, where most influenza virus subtypes replicate in humans. However, in this study, we did not test the intranasal administration of aprotinin. Future studies will have to be performed to determine the optimal dosage and route of administration for human application. Additionally, whether aprotinin will be effective against highly pathogenic avian influenza viruses (HPAIVs) will have to be evaluated. HPAIVs have multibasic cleavage sites that are more accessible to a wide range of proteases.[35] If aprotinin has the ability to inhibit HPAIVs, then it will be a viable pandemic influenza therapeutic candidate that runs a lower risk of causing drug resistance than currently used antivirals like oseltamivir.
In this study, we were able to demonstrate that aprotinin inhibitsin vitro production of 1) avian IAVs with zoonotic potential, 2) oseltamivir-resistant IAV, and 3) currently circulating IBV, which is inherently less susceptible to oseltamivir. We propose that aprotinin is an excellent candidate for the treatment of most IAVs and IBVs in humans. However, whether aprotinin is similarly effective against HPAIVs together with the selection of the most appropriate route of administration and the optimal dosage for its clinical use still needs to be determined in future studies.