Elsevier

Antiviral Research

Volume 92, Issue 1, October 2011, Pages 27-36
Antiviral Research

Aprotinin and similar protease inhibitors as drugs against influenza

https://doi.org/10.1016/j.antiviral.2011.07.014Get rights and content

Abstract

Efforts to develop new antiviral chemotherapeutic approaches are focusing on compounds that target either influenza virus replication itself or host factor(s) that are critical to influenza replication. Host protease mediated influenza hemagglutinin (HA) cleavage is critical for activation of virus infectivity and as such is a chemotherapeutic target. Influenza pathogenesis involves a “vicious cycle” in which host proteases activate progeny virus which in turn amplifies replication and stimulates further protease activities which may be detrimental to the infected host. Aprotinin, a 58 amino acid polypeptide purified from bovine lung that is one of a family of host-targeted antivirals that inhibit serine proteases responsible for influenza virus activation. This drug and similar agents, such as leupeptin and camostat, suppress virus HA cleavage and limit reproduction of human and avian influenza viruses with a single arginine in the HA cleavage site. Site-directed structural modifications of aprotinin are possible to increase its intracellular targeting of cleavage of highly virulent H5 and H7 hemagglutinins possessing multi-arginine/lysine cleavage site. An additional mechanism of action for serine protease inhibitors is to target a number of host mediators of inflammation and down regulate their levels in virus-infected hosts. Aprotinin is a generic drug approved for intravenous use in humans to treat pancreatitis and limit post-operative bleeding. As an antiinfluenzal compound, aprotinin might be delivered by two routes: (i) a small-particle aerosol has been approved in Russia for local respiratory application in mild-to-moderate influenza and (ii) a proposed intravenous administration for severe influenza to provide both an antiviral effect and a decrease in systemic pathology and inflammation.

Introduction

Emergence of the novel “swine”-origin influenza H1N1 (H1N1pdm) pandemic stresses the need for the development of new antiviral drugs. Only two classes of specific antiviral compounds are currently in use in medical practice against influenza. These are amantadine derivatives (Symmetrel and Flumadin), which specifically block the influenza A virus ionic channel M2 (Pinto and Lamb, 2006), and oseltamivir (Tamiflu) and zanamivir (Relenza), which specifically suppress influenza A and B virus neuraminidases (Moscona, 2005). Unfortunately, emergence and spread of virus variants resistant to both M2- and NA-inhibitor drugs have been documented in influenza H1N1, H3N2, H1N1pdm, and H5N1 virus-infected patients (CDC, 2011, Moscona, 2009, Thorlund et al., 2011). The emergence of resistance without a major loss of virus fitness contributes to the need for alternative antivirals to combat modern epidemics of influenza. Research efforts are focusing on development of new antiviral chemotherapeutic approaches that target influenza virus replication itself or host factor(s) which are integral to virus replication, or interruption of virus-mediated pathogenesis in the host.

These chemotherapeutic principles are referred to as virus- and host-targeted antivirals. Virus-targeted agents inhibiting protein HA (Russell et al., 2008), NS1 (Walkiewicz et al., 2011), NP (Kao et al., 2010), polymerase proteins (Furuta et al., 2005, Ghanem et al., 2007), viral RNA survival (Wu et al., 2008) are in development. Host-targeted virus inhibitors include the NF-κB and Raf/MEK/ERK pathways (Ludwig, 2009), the ubiquitination process (Liao et al., 2010), expression of cellular sialic acid receptors (Belser et al., 2007, Malakhov et al., 2006, Zhang, 2008) and protease inhibitors – the subject of this review. A theoretical advantage of a host targeted antivirals, including protease inhibitors, is that resistance will not be selected for as readily as it is by influenza virus for anti-viral targets.

Of the protease inhibitors, there has been the greatest experience with aprotinin. Aprotinin is a serine protease inhibitor that effectively targets host trypsin-like protease(s) responsible for influenza virus hemagglutinin cleavage and virus activation in infected organism (Zhirnov, 1983). This anti-protease compound has a long history in medical practice in the treatment of pancreatitis and post-operative bleeding (Fritz and Wunderer, 1983, Trautschold et al., 1967). There are several licensed compounds of aprotinin, including Trasylol™ (Bayer AG, Germany), AntagosanTM (Sanofi Aventis, France), GordoxTM (Gedeon Richter, Hungary), and ContrycalTM (AWD, Germany). There is an influenza specific approval for topical use of aprotinin in Russia. Evaluation of aprotinin as a representative of the protease-inhibitor class of host-targeted anti-influenzal agents will be highlighted in this minireview.

Section snippets

Influenza virus proteolytic activation and host proteases involved in this process

More than 30 years ago it was shown that influenza viruses require activation by host specific protease(s) (influenza activating protease – IAP) to establish productive multicycle replication in tissue culture (Klenk et al., 1975, Lazarowitz and Choppin, 1975). This activation process is mediated by cleavage at a specific site of the virus hemagglutinin protein (HA). HA assembles as a homotrimer forming external spikes on the surface of the virion (Wilson et al., 1981). HA is synthesized as a

Aprotinin targets influenza HA0 cleavage and suppresses multicycle virus replication

Aprotinin is the model protease inhibitor that has progressed the furthest in preclinical and clinical evaluation. One of the characteristics of aprotinin is its high general tolerance even at large dosages; for example, the LD50 in mice is 2.5 × 106 Kallikrein Inhibiting Units (KIU)/kg and in dogs intravenous administration of 1.0 × 106 KIU/kg is tolerated without complications (for review see (Trautschold et al., 1967)). It is a single-chain globular polypeptide isolated from bovine lung tissue.

Benefits and precautions in aprotinin usage against influenza

The preclinical efficacy of aprotinin against influenza in vitro and in vivo experiments prompted us to study aprotinin therapy in humans with influenza and influenza-like infections. Since influenza infection primarily locates in the upper-middle respiratory tract of humans, small-particle aerosol inhalations were utilized. A clinical study was conducted during a seasonal outbreak of acute respiratory illness caused predominantly by influenza and parainfluenza viruses (January–May 1994) (

General conclusions

Host trypsin-like proteases are key factors in facilitating influenza virus infection because they cleave virus HA glycoprotein to trigger its cell fusogenic activity and activate virus infectivity with promotion of infection. In turn, influenza virus directly damages airway epithelium and stimulates host proteases, which mediate development of inflammation in the infection locus. Aprotinin, a polyvalent protease inhibitor from bovine lung, was found to suppress influenza virus proteolytic

Disclosure statement

The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript. OPZ has US, European, Japan, and Russian patents disclosing the use of aerosolized aprotinin for the treatment of influenza. Other authors declare no competing interests.

Acknowledgments

Funding was provided by the Russian Foundation of Basic Research (Grant 10-04-01824), the German SFB 593 program, and a NATO collaborative grant HT-974619. Authors thank Olga and Veronica Zhirnova for the help in preparation of figures.

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    These data have been partially presented at the Antivirals Congress – Celebrating 30 years of the Journal Antiviral Research. Abstr. P1-55 entitled: “Proteolytic activation and multicycle replication of pandemic swine influenza virus H1N1 are inhibited by aprotinin” by O.P. Zhirnov, T. Matrosovich, M. Matrosovich, and H.D. Klenk, November 7–9, 2010; Amsterdam, The Netherlands.

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