Antioxidant Therapy as a Potential Approach to Severe Influenza-Associated Complications

With the appearance of the novel influenza A (H1N1) virus 2009 strain we have experienced a new influenza pandemic and many patients have died from severe complications associated with this pandemic despite receiving intensive care. This suggests that a definitive medical treatment for severe influenza-associated complications has not yet been established. Many studies have shown that superoxide anion produced by macrophages infiltrated into the virus-infected organs is implicated in the development of severe influenza-associated complications. Selected antioxidants, such as pyrrolidine dithiocabamate, N-acetyl-l-cysteine, glutathione, nordihydroguaiaretic acid, thujaplicin, resveratrol, (+)-vitisin A, ambroxol, ascorbic acid, 5,7,4-trihydroxy-8-methoxyflavone, catechins, quercetin 3-rhamnoside, iso- quercetin and oligonol, inhibit the proliferation of influenza virus and scavenge superoxide anion. The combination of antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections. These results suggest that an agent with antiviral and antioxidant activities could be a drug of choice for the treatment of patients with severe influenza-associated complications. This review article updates knowledge of antioxidant therapy as a potential approach to severe influenza-associated complications.


Introduction
With the appearance of the novel influenza A (H1N1) virus 2009 strain we have recently experienced a new influenza pandemic [1,2]. The clinical spectrum of pandemic influenza A (H1N1) virus infection was broad, ranging from mild upper respiratory tract illness with or without fever and occasional gastrointestinal symptoms such as vomiting or diarrhea and exacerbation of underlying conditions, to severe complications such as pneumonia resulting in respiratory failure, acute respiratory distress syndrome, multi-organ failure and even death [3,4].
Many patients died from severe complications associated with the pandemic influenza A (H1N1) virus infection despite receiving intensive care [3,4], and as of the 25 th of July 2010, 18,398 laboratory-confirmed fatal cases of pandemic influenza A (H1N1) have been reported to the World Health Organization [5]. The influenza A (H1N1) virus human infections event has now moved into a post-pandemic period, with a pattern that has been transitioning towards that of seasonal influenza [6]. Beside the influenza A (H1N1) pandemic the global burden of seasonal influenza epidemics is believed to be some 3-5 million cases of severe illness and 300,000-500,000 deaths every year [7]. Additionally, we still face the threat of infection with the highly pathogenic avian influenza A (H5N1) virus.
Three classes of anti-influenza drugs have been used for chemoprophylaxis and treatment of influenza virus infections [8] (Figure 1): amantadine (1) and rimantadine (2) which inhibit viral membrane protein (M2) of the proton channel that is necessary for uncoating; oseltamivir (3), zanamivir (4), peramivir (5) and laninamivir octanoate (6) which inhibit viral neuraminidase (NA) that is necessary for virion release and ribavirin (7) that inhibits enzyme activity essential for viral replication. Initial diagnostic testing found that the pandemic influenza A (H1N1) virus was susceptible to NA inhibitors, but resistant to M2 inhibitors [9], therefore, oseltamivir has been used widely for treatment and chemoprophylaxis of pandemic influenza A (H1N1) [2].
Sporadic cases of oseltamivir-resistant pandemic influenza A (H1N1) virus have been reported worldwide [10]. This oseltamivir resistance was caused by the NA mutation H275Y [10].
Person-to-person transmission of oseltamivir-resistant viruses in healthy adults has been confirmed [11]. In cases of development of oseltamivir-resistance, treatment options are limited because zanamivir is not licensed for treatment of children under 7 years old and is contraindicated in persons with underlying airway disease. Recently, it has been reported that a single inhalation of laninamivir octanoate was an effective and well-tolerated drug for the treatment of children with oseltamivir-resistant influenza A (H1N1) virus infection [12].
Additionally, intravenous drip infusion of peramivir has offered a new treatment option for children and infants suffering from influenza virus infections and patients where oral administration was difficult or not possible [13]. It was also effective for severe influenza-associated complications, such as acute respiratory failure [14]. Nonetheless, NA inhibitor-resistant viruses with H275Y mutation emerged early and replicated in patients, who have received hematopoietic cell transplant, under treatment with immunosuppressive drugs after intravenous drip infusion of peramivir [15]. A young adult with pandemic influenza A (H1N1) virus infection was treated with intravenous peramivir, but died from severe viral pneumonia [16]. These results suggest the need for development of new anti-influenza drugs utilizing alternative antiviral mechanisms and consideration of using anti-influenza drug combinations. Some such approaches have been explored, whereby a triple combination of amantadine, ribavirin and oseltamivir was highly active and synergistic against drug resistant influenza virus strains in vitro [17].    In cases of severe influenza-associated complications, the pathological manifestations are the   result of complex biological phenomena, such as apoptosis induction, macrophage activation, oxidative tissue damage and higher contents of pro-inflammatory cytokines [18]. The pathogenesis of severe influenza-associated complications involves not only apoptotic cell death mediated through virus replication in the infected cells, but also the injury of non-infected cells by superoxide anion derived from activated phagocytes (i.e., macrophages and neutrophils) infiltrated into the virus-infected organs [19]. As illustrated in Figure     complexes, and rapid transport via a lipophilic complex by PDTC has been proposed to explain the intracellular recruitment of copper and zinc ions from the extracellular medium [39]. It has been demonstrated that copper and zinc ions inhibit influenza virus RNA-dependent RNA polymerase activity, and that the inhibitory effect of bathocuproine-copper and bathocuproine-zinc complexes is greater than that of bathocuproine itself [40]. Moreover, it has been known that PDTC-copper and PDTC-zinc complexes (9 and 10, respectively, Figure 3) inhibited the replication of Coxsackie virus [41] and rhinovirus [42].

N-Acetyl-L-cysteine
N-Acetyl-L-cysteine (NAC, 11, Figure 3), the acetylated derivative of the amino acid L-cysteine, is an excellent source of thiol groups, and is converted in the body into metabolites capable of stimulating glutathione synthesis, promoting detoxification, and acting directly as free radical scavengers [45]. NAC inhibited the induction of apoptosis [46][47][48] and gene expression for pro-inflammatory cytokines and chemokines such as IL-6, IL-8, RANTES and interferon-inducing protein (IP)-10, by influenza virus infection [48]. NAC inhibited the proliferation of influenza virus at an early, but not later, stage of infection [47,48].
Administration of NAC significantly decreased the mortality in mice infected with influenza virus [49], and a combination of NAC and ribavirin synergistically reduced the lethal effects [50]. Furthermore, a combination of NAC and oseltamivir also synergistically reduced the lethal effect of influenza virus infection in mice [51]. These results support the notion that combinations of antioxidant therapy with current drugs can improve the treatment of influenza virus infections.
Administration of NAC appears to reduce symptomatic conditions associated with influenza virus infection. A total of 262 subjects of both sexes were given either placebo or NAC (600 mg) orally twice daily for six months. Although incidents of seroconversion towards A (H1N1) Singapore 6/86 influenza virus was similar in the two groups, NAC treatment decreased both the incidence and severity of influenza-like episodes, and the length of time confined to bed. The authors concluded that NAC did not prevent influenza A (H1N1) virus infection, but did significantly reduce the incidence of clinically apparent disease [52]. In another paper, it has been reported that a patient with viral pneumonia caused by the novel influenza A (H1N1) virus 2009 infection and septic shock improved rapidly after continuous intravenous infusion of high-dose NAC at 100 mg/kg combined with oseltamivir [53].
The scavenges oxygen radicals, such as peroxynitrite, singlet oxygen, hydroxyl and superoxide anion radicals [56]. The ability of NDGA to scavenge the above-mentioned oxygen radicals is much greater than that of reference tested compounds, such as uric acid, penicillamine, reduced glutathione and mannitol [56]. These strong antioxidant properties may be due to the presence of four reducing equivalents from the two catechol groups in NDGA; hydrogen atoms of the four phenolic hydroxyl groups react with oxygen radicals [56]. NDGA has been shown to have promising applications in the treatment of multiple diseases, including cardiovascular diseases, neurological disorders, cancers and virus infections [57].

Terameprocol
NDGA derivatives did not affect the expression of reporter genes driven by the adenovirus major late promoter and the cytomegalovirus promoter [61]. It is predicted that the antiviral activity of NDGA derivatives is selective, depending on the virus types. Influenza virus has viral RNA-dependent RNA polymerases, which contribute to the replication and transcription processes of the viral genes, probably irrespective of cellular transcription factor Sp-1. An additional mechanism of NDGA for the inhibition of influenza virus proliferation has been proposed. NDGA is shown to inhibit the intracellular transport of vesicular stomatitis virus glycoproteins [62]. Conceivably, NDGA may inhibit influenza virus proliferation via inhibition of intracellular transport of viral glycoproteins.

Resveratrol
The plant polyphenol resveratorol (3,5,4'-trihydroxy-trans-stilbene) (16, Figure 4) inhibited the progressive effects of superoxide anion and hydrogen peroxide radicals on arachidonic acid production and cyclooxygenase-2 induction in macrophages [66]. Resveratrol inhibited the replication of influenza virus in MDCK cells, as a result of the blockade of the nuclearcytoplasmic translocation of viral ribonucleoproteins and the reduced expression of late viral protein, such as HA and matrix protein [67]. Resveratol also improved survival and decreased pulmonary viral titers in influenza virus-infected mice [67]. Since resveratrol inhibited the induction of RANTES production by influenza virus infection in A549 lung epithelial cells [68], it is presumed that resveratorol attenuate the activation of macrophages during influenza virus infection.
(+)-Vitisin A (17, Figure 4), a tetramer of resveratrol, isolated from Vitis thunbergii was reported to have various bioactivities, including antioxidant, protection against platelet aggregation, to inhibit the biosynthesis of pro-inflammatory cytokine leukotriene B4, and to suppress TNF-α-induced monocyte chemoattractant protein production in primary human endothelial cells [69][70][71]. Furthermore, (+)-vitisin A has been shown to inhibit the production of RANTES in airway epithelial cells after influenza A virus infection, the effect of which was much higher than that of resveratrol [72]. Accordingly, it has been suggested that (+)-vitisin A may serve as a potential anti-inflammatory agent that interrupts the pathogenesis after viral infection.

Ambroxol
Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine, 18, Figure 4), known as a mucolytic agent, has been used for the treatment of chronic bronchitis and neonatal respiration distress syndrome [73]. Ambroxol suppressed the proliferation of influenza virus in the mouse airway and improved the survival rate of mice [74]. Antioxidant activity of ambroxol is related to the direct scavenging effect for ROS, such as superoxide anion and hydroxyl radicals [75,76]. Treatment with ambroxol also significantly decreased the incidence of acute upper respiratory diseases during winter season in humans [77].

Ascorbic acid
Ascorbic acid (19, Figure 4) scavenges superoxide anion [78]. Ascorbic acid inhibited the proliferation of influenza virus in cell cultures [79]. Dehydroascorbic acid, an oxidized form of ascorbic acid without reducing ability, showed much stronger antiviral activity than that of ascorbic acid, indicating that the antiviral activity of ascorbic acid is due to factors other than antioxidant mechanism [80]. In a controlled trial of 226 patients with influenza A, 114 patients received vitamin C 300 mg/day, and 112 patients served as controls; outcomes measured were development of pneumonia and duration of hospital stay. Pneumonia was reported in two subjects in the treatment group and 10 in the control group, and hospital stays for influenza or related complications averaged nine days in the vitamin C group and 12 days in the control group [81]. Therefore it has been considered that combined inhalation and oral supplementation of ascorbic acid may prevent influenza virus infection [82].

Flavonoids
Flavonoids are a ubiquitous group of polyphenolic substances which are present in most plants, concentrating in seeds, fruit skin or peel, bark, and flowers. The structural components common to these molecules include two benzene rings on either side of a 3-carbon ring.  Figure 5) isolated from the roots of Scutellaria baicalensis, was shown to have a specific inhibitory activity against influenza virus NA because it did not affect the mouse liver NA activity [84,85]. F36 inhibited the proliferation of influenza virus in MDCK cells, in the allantoic sac of embryonal chicken egg and in vivo using BALB/c mice [85][86][87]. Immunoelectron microscopic analysis revealed that F36 inhibited the budding of progeny influenza virus particles from MDCK cell surface and microvilli [88].

Catechins
Catechins from green tea: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC) (21, 22 and 23, respectively, Figure 5) have been evaluated for their ability to inhibit influenza virus replication in cell culture [89]. Among the test compounds, EGCG and ECG were found to be potent inhibitors of influenza virus replication in MDCK cell culture, and this effect was observed in all influenza virus subtypes tested, including A (H1N1), A (H3N2) and B virus. The 50% effective inhibition concentrations of EGCG, ECG, and EGC for influenza A virus were 22-28, 22-40 and 309-318 µM, respectively. EGCG and ECG exhibited inhibitory activity of hemagglutination, suppressed viral RNA synthesis in MDCK cells, and inhibited the NA activity, however, the effects of EGC were much lesser. The results show that the 3-galloyl group of catechin skeleton plays an important role on the observed antiviral activity, whereas the 5'-OH at the trihydroxybenzyl moiety at the 2-position plays a minor role. Catechins have been shown to possess the ability to scavenge for superoxide anion and hydroxyl radicals [90]. Gargling with tea catechin extracts prevented influenza virus infection in elderly nursing home residents [91]. Long chain monoester derivatives of EGCG enhanced the anti-influenza virus activity 24-fold relative to native EGCG [92].  The influenza A RNA polymerase possesses endonuclease activity to digest the host mRNA.
This endonuclease domain can be a target of anti-influenza A virus drug. Kizuhara and co-workers have reported that green tea catechins inhibited this viral endonuclease activity and that their galloyl group was important for their function [93]. Docking simulations revealed that catechins with galloyl group fitted well into the active pocket of the endonuclease domain to enable stable binding. Their results provide useful data that make it possible to refine and optimize catechin-based drug design more readily for stability.

Quercetin 3-rhamnoside
Quercetin 3-rhamnoside (Q3R, 24, Figure 5) from Houttuynia cordata possessed strong antiviral activity against influenza A/WS/33 virus as well as oseltamivir [94]. Pre-exposure of the virus to Q3R did not alter the infectivity. When Q3R was added just after the virus infection or until four hours after the virus infection, the antiviral activity of Q3R was exhibited. Viral mRNA synthesis was inhibited by the treatment with Q3R. The mode of action of Q3R involved the inhibition of virus replication in the initial stage of virus infection by indirect interaction with virus particles.

Isoquercetin
Isoquercetin (25, Figure 5) inhibited the replication of both influenza A and B viruses in cell cultures, the antiviral activity of which was much stronger than that of EGCG, resveratrol and quercetin [95].