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.