**5. Vitamin E in the influenza therapy**

Currently, treatment of influenza is directed mainly at targeting the first pathogenetic component through administration of specific antivirals. Application of correctors of influenza pathogenesis that are associated with controlling inflammation and oxidative stress remains in the background.

Among the antioxidants tested in influenza virus infections in mice [17, 49–51, 65], α-tocopherol (vitamin E) occupies the leading position because of its efficacy in preventing oxidative damage through its free-radical scavenging activity [16, 18, 24, 42, 53–55, 66, 67].

Although vitamin E is not an agent with specific antiviral action, its antioxidant effect probably plays an important role in liver protection.

The most important question is whether vitamin E, as a natural antioxidant, could be used as anti-influenza agent.

In fact, the ideal protective agent against flu should fulfill several criteria: (a) it must not allow the formation of resistant viral strains; (b) it must have a general protective effect on the majority of organs; (c) it must have an acceptable toxicity profile and protective time-window effect; and especially importantly, (d) it must provide strong protection against the symptoms of emerging influenza, such as the oxidative state of the infected body.

Evidently, a more effective treatment strategy is needed. Immunomodulators have been proven to be highly successful in treating the flu, at least in mouse infection models [68–71]. Using antioxidative agents to act directly on downstream deleterious inflammation events is also of significant importance in flu therapy.

The preventive effects of vitamin E and vitamin C, alone and in combination, was tested on the damage caused by influenza virus infection [72]. Mice, infected with influenza virus A/2/68/(H3N2) (1.5 LD50), were administered once-daily doses of vitamin E (60 mg/kg b.w.) and vitamin C (80 mg/kg b.w.) intraperitoneally (for 3 days before virus inoculation). Vitamin E effectively restored lipid peroxidation levels increased by influenza virus infection. The effect of vitamin C was similar, but slighter. The combination (vitamin E + C) had a greater effect on lipid peroxidation levels than did their separate administration. P-450-dependent monooxygenase activity was significantly restored, and more pronounced cytochrome P-450 content and NADPH-dependent cytochrome *c* reductase activity was noted. The preventive effect of vitamin E was stronger than that of vitamin C, but the combination (vitamin E + C) had the strongest effect. The superior protective effect of the combination is probably due to the better interaction between hydrophobic and hydrophilic low-molecular-weight antioxidants against a free-radical disease like influenza. The mechanism of this interaction is related to vitamin C's ability (when situated in aqueous phase) to recycle vitamin E (located in membranes), repairing vitamin E's tocopheroxyl radical. Thus, vitamin C promotes the function of vitamin E as a free-radical scavenger [73, 74].

However, researchers have shown that endogenic levels of vitamin E are significantly decreased in lung, liver, and blood plasma (**Table 1**) during the course of flu infection [21, 23, 24, 42]. Animal and human studies have demonstrated a negative correlation between endogenous levels of vitamin E in the body and pulmonary inflammations, and exogenous vitamin E supplementation has been tied to reducing severe symptoms of lung disease [16, 18, 64].

Currently, treatment of influenza is directed mainly at targeting the first pathogenetic component through administration of specific antivirals. Application of correctors of influenza pathogenesis that are associated with controlling inflammation and oxidative stress remains

Among the antioxidants tested in influenza virus infections in mice [17, 49–51, 65], α-tocopherol (vitamin E) occupies the leading position because of its efficacy in preventing oxidative dam-

Although vitamin E is not an agent with specific antiviral action, its antioxidant effect prob-

The most important question is whether vitamin E, as a natural antioxidant, could be used as

In fact, the ideal protective agent against flu should fulfill several criteria: (a) it must not allow the formation of resistant viral strains; (b) it must have a general protective effect on the majority of organs; (c) it must have an acceptable toxicity profile and protective time-window effect; and especially importantly, (d) it must provide strong protection against the symptoms

Evidently, a more effective treatment strategy is needed. Immunomodulators have been proven to be highly successful in treating the flu, at least in mouse infection models [68–71]. Using antioxidative agents to act directly on downstream deleterious inflammation events is

The preventive effects of vitamin E and vitamin C, alone and in combination, was tested on the damage caused by influenza virus infection [72]. Mice, infected with influenza virus A/2/68/(H3N2) (1.5 LD50), were administered once-daily doses of vitamin E (60 mg/kg b.w.) and vitamin C (80 mg/kg b.w.) intraperitoneally (for 3 days before virus inoculation). Vitamin E effectively restored lipid peroxidation levels increased by influenza virus infection. The effect of vitamin C was similar, but slighter. The combination (vitamin E + C) had a greater effect on lipid peroxidation levels than did their separate administration. P-450-dependent monooxygenase activity was significantly restored, and more pronounced cytochrome P-450 content and NADPH-dependent cytochrome *c* reductase activity was noted. The preventive effect of vitamin E was stronger than that of vitamin C, but the combination (vitamin E + C)

It is well known that vitamin E is able to prevent oxidative damages [58–60].

age through its free-radical scavenging activity [16, 18, 24, 42, 53–55, 66, 67].

of emerging influenza, such as the oxidative state of the infected body.

**5. Vitamin E in the influenza therapy**

ably plays an important role in liver protection.

also of significant importance in flu therapy.

in the background.

74 Vitamin E in Health and Disease

anti-influenza agent.

An underappreciated approach in flu therapy continues to be combination administration regimens of specific viral replication inhibitors together with antioxidants. Therefore, investigations on the combination effects of specific anti-influenza chemotherapeutic agents and antioxidants are of special interest. Previously, we established a favorable combination effect of the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol) with M2-blocker rimantadine in mice infected with influenza virus A(H3N2). Ionol was administered intraperitoneally in a 3-day course (45 or 75 mg/kg daily) before virus inoculation, and rimantadine (oral application of 15 mg/kg) was administered for 5 days following the day of infection [75].

Recently, a strong beneficial effect of the combination of α-tocopherol (a component of vitamin E) and oseltamivir was demonstrated in the treatment of experimental infection with influenza virus A/H3N2 in mice [76]. The results showed that this combination of agents simultaneously suppressed the two main processes in the pathogenesis of influenza—the development of pulmonary lesions in the respiratory tract as a result of virus replication and the oxidative stress damage to membranes of small vessels and other tissues in the body thus characterizing it as a very good prospect for flu therapy.

However, a question arose: Is oseltamivir an antioxidant? We used some model systems to test oseltamivir's ability to scavenge superoxide radicals, to inhibit their generation, and to influence Fe2+ or (Fe2+-EDTA)-induced lipid peroxidation in liposomal egg suspension and in lung and liver microsomes [77]. We concluded that the reduction of oxidative stress in vivo is not connected with oseltamivir's effect on the development of free-radical processes in the organism. Oseltamivir's effect on oxidative stress in the course of viral infection could be explained by its specific therapeutic effect, which is connected with suppression of viral replication in the target organ.

The in vivo antiviral activity of the combination vitamin E + oseltamivir, expressed by a marked protective effect on the survival of influenza A virus-infected animals, was recorded when vitamin E was administered simultaneously with oseltamivir phosphate via a 5-day course post virus inoculation [76]. This effect was not observed when the vitamin E course started 120 or 48 hours before viral inoculation. According to this study, vitamin E applied individually had no effect on the course of influenza A virus infection caused by 10 MLD50. Only a lower value of the lung index was registered. In our previous study, we established a protective effect of vitamin E at virus infection with 2 MLD50 [18, 24].

Special attention should be paid to the sharp synergistic character of the antiviral effect of the combination vitamin E and oseltamivir at a dose of 0.625 mg/kg administered simultaneously, which resulted in the following: (i) a pronounced increase in the protection index, attaining 76%, and a lengthening of the MSD by 3.2 and 4 days; (ii) a pronounced decrease in lung infectious virus titer; and (iii) a strong reduction in lung lesions. Oseltamivir at the same dose applied separately did not manifest antiviral activity.

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The observed phenomenon of a strong oseltamivir dose-dependence of the combined anti-flu effect attaining a pronounced synergism at the lowest tested dose of 0.625 mg/kg merits a special attention. One explanation for this phenomenon could be the interaction of these two agents related to their specific mechanisms of action on the viral target structures in the lung. It is well known that vitamin E is included in the cellular lipid bilayer, thus decreasing cellular membrane permeability [60, 62, 64]. Oseltamivir, for its part, mimics cellular neuraminic acid, thus interfering with the exit process of the new progeny virions [78, 79]. These two processes run in parallel, thus opposing the virus infection course on the cellular level. The two substances most likely compete in the modification of cellular membranes through their specific mechanisms of action. Therefore, the place of emerging synergism for oseltamivir and vitamin E is most likely the cell membrane in the viral target area in the lung.

Тhe favorable (even synergistic) type оf interaction between vitamin E and oseltamivir was absent when vitamin E was administered for 5 days before virus inoculation—that is, before the onset of the oseltamivir course.

The described results suggest that vitamin E has an important place as a component of the complex therapy of epidemic flu when administered simultaneously with chemotherapeutic agents, such as neuraminidase inhibitors. Moreover, in addition to its membrane protective effect in the influenza virus target area, vitamin E manifests pronounced activities as an antioxidant agent and as a protein kinase C inhibitor and a protector of lung tissue during inflammatory lung illnesses [59, 80]. The study discussed above [76] convincingly demonstrates a strong beneficial effect of the combination of vitamin E and oseltamivir in the treatment of experimental infection with influenza virus A/H3N2 in mice.

The results show that this combination of agents simultaneously suppresses the two main processes in the pathogenesis of influenza, the development of pulmonary lesions in the respiratory tract resulting from virus replication and the oxidative stress damage to the membranes of small vessels and other tissues in the body, thus characterizing it as a very likely prospect in the therapy of flu.

In summary, vitamin E could be recommended as a reliable agent, a component in multitarget influenza therapy.
