Introduction

Antiviral drugs substances exhibiting interference with individual replication stages viruses are a need to search because of the limited number of them. The conventional “one virus – one drug” model actually makes the most of the possibilities therapeutic. Searching can therefore be one of the strategies for controlling viral infections takes into account pleiotropic effect and strengthens the antiviral reaction it also includes substances modulating the immune system.⁽¹⁾

Interferon (IFN) is a cytokine involved in the body’s immune response at sites and at systemic level, and involved in reducing infection viral ⁽²⁾. IFN was described in 1957 by Issacs and Lindemann as a factor that interferes with viral replication, often the most common protective one the impact of interference on herpes infection of the set as early as the 1930s as so-called Margassi phenomenon.⁽³⁾ Produced by a cell of the immune system IFN-α includes pleiotropic antiviral activity. It binds to specific ones receptors for cells and analysis based on antiviral proteins. Viral dsRNA is an inducer of IFN-α production. In infected cells antiviral effect of interferon generated with enzyme activity: 2′, 5′ -oligoadenyl synthetase and protein kinase R(PKR), resulting in inhibition of synthesis proteins by PKR phosphorylation and degradation of viral ⁽⁴⁾. IFN-α stimulates also cellular response by activating effector cards. Stimulates for example, pre-NK cells for differentiation into NK cells and activates early, natural protection against infection. INF-α together with IFN-α increase expression MHC class I molecules, which improves the ability of the cell to present antigen. State antiviral, stimulated by the presence of IFN-α in the infected cell, lasts about 2-3 days.⁽⁵⁾ Despite the fact that about 60 antiviral drugs are currently available, close half of them are registered for the treatment of HIV infection. Others are used in the treatment of viral hepatitis (HBV, HCV), human infections herpesviruses and influenza viruses. There is still a lack of possibilities to control many important ones clinically and epidemiologically viral infections ⁽⁶⁾. Infections caused by adenoviruses or parainfluenza viruses are common, usually mild, self-limiting infections. However, the immune system can cause serious complications. Adenoviruses initially infect or reactivate in 20-50% of people with immunosuppression, causing organ or generalized infections.⁽⁷⁾ Parainfluenza viruses in situations of weakened immune mechanisms the body cause acute pneumonia, requiring artificial ventilation and in part cases (5-35%) resulting in death.⁽⁸⁾ None of the previously known preparations antiviral has no registration for the treatment of infections with these viruses. Isoprinosine is a complex of inosine, p-acetamidobenzoate and diamidopropanol – a drug belonging to it to the group of general purpose antiviral pharmaceuticals.⁽⁹⁾ Isoprinosine except antiviral activity has confirmed in both in vitro and in clinical trials, an immunomodulatory effect. ⁽¹⁰⁾ It is likely that this compound is involved in inhibition viral RNA synthesis. Stimulates the immune system affecting puberty as well activation of lymphocytes. Numerous studies (in vitro and in vivo) have been shown to increase Isoprinosine production of cytokines such as interleukin (IL-1,IL-2 and IL-12), interferon α (IFN-α),  TNF-α and interferon γ (IFN-γ), inhibits IL-10 production, enhances the effect cytotoxic (natural killer; NK cells) and stimulates chemotaxis and phagocytosis.⁽¹¹⁾ The aim of the study was to assess the effect of interferon-α and Isoprinosine on titers infectious RNA viruses: parainfluenza viruses (human parainfluenza 2 virus; HPIV-2) and human C adenoviruses(human adenovirus 2; HAdV-2) in vitro.

Material and Methods

RNA viruses pathogenic to humans were used in the study: adenovirus 2 (HAdV-2) and parainfluenza virus type 2 (HPIV-2). Viruses propagated in A549 cell line (ATCC^® CCL-185MT). Cells were cultured in medium Dulbecco’s Modified Eagle’s Medium (DMEM, ThermoFisher Scientific) supplemented with 10% calf fetal bovine serum (FBS; ThermoFisher Scientific) and a 1% penicillin mixture /streptomycin and antimicotic solution (BI; Biological Industries) and 5% atmosphere . For reproduction viruses, and evaluation of antiviral activity, reduced fluid medium was used up to 2% FBS concentration. Interferon-α (Switzerland) was added to the culture at concentrations of 100 and 1000 IU/ml. Interferon doses were selected based on literature analysis and own experience. Isoprinosine (BI; Biological Industries) in a dose 5.0 mg were suspended in 5.0 mL of Phosphate-buffered saline (PBS,PH=6.9), filtered using a millipore filter (Filter Unit, 0.2 μm). Prepared non-toxic for cell culture concentration: 50-800 μg / ml. The selection of doses was determined on the basis of previously conducted and published research results, in which using the method qualitative using an inverted optical microscope (OLYMPUS), image enlarged by 400x and by quantitative method using the tetrazole salt reduction test in cell mitochondria, MTT Cell Proliferation Assay (ATCC bioproducts ™, USA) has been shown to be non-toxic isoprinosine activity on A549 culture , HEp-2 and HEL 299 cell lines.⁽¹²⁾ Antiviral activity testing involved infection of A549 cells (1×105 cells / ml) with each virus at a dose of 100 TCID₅₀ / ml (Tissue Culture Infectious Dose). After 60 min incubation of the virus with cells in micro plates (in a volume of 0.2 ml in flat-bottom 96-well plates, (MEDLAB-PRODUCTS) twice cultures were washed with PBS to remove non-cell-associated viruses. Then, infected cells were added:

D-MEM medium (virus control).

Isoprinosine at concentrations from 50 to 800 μg / ml in DMEM liquid medium.

IFN-α at a concentration of 100 or 1000 IU / ml.

Isoprinosine and IFN-α in w/w concentrations.

The exposure time in each system was 48 hours the antiviral effect was determined by the method of reduction of infectious titers (YRA – yield reduction assay). The virus infectious titer is expressed in log10 TCID₅₀ / ml. Significance was determined differences between average virus titers, cell culture microplates were frozen and thawed three times (to release viruses from cells), centrifuged for 10 minutes (3000 rpm, temperature 4 °C). The supernatant was used to assess viral load according to the Reed-Muench method.⁽¹³⁾

Statistical Analysis

Student’s t-test was used for related variables, the significance level P <0.05 was adopted. The results were statistically evaluated using the Pearson correlation method measuring the relationship between isoprinosine doses and viral load and INF-α. Correlation coefficient value in the range of 0.4-0.7 interpreted as a moderate relationship, 0.7-0.9 relationship significant above 0.9 as a very strong relationship.

Results

There was no toxicity to Isoprinosine at concentrations between 50 and 800 μg / ml for culturing. IFN-α has been shown to significantly inhibit the multiplication of these viruses in research in vitro (P <0.05). The reduction of infectious titers was linearly dependent on concentration IFN-α. The concentration of 100 IU / ml , 1000 IU / ml and isoprinosine in concentrations 200μg / ml and 800μg / ml. IFN-α reduced the infectious titre HPIV-2, and HAdV-2 respectively by more strongly inhibited replication resulting in 100 IU / ml , 1000 IU / ml and isoprinosine in concentrations 50μg / ml, 100 μg / ml and 400μg / ml compared with a control. Analyzing the Pearson correlation coefficient was demonstrated significant (P <0.05) dependence of the infectious titre HPIV-2 and HAdV-2 on the dose of isoprinosine in cultures A549. Similarly, a high correlation coefficient (however not statistically significant) was calculated for other RNA viruses: HPIV-2and HAdV-2. isoprinosine most strongly reduced HAdV-2 infectious titers compared to control , but increased isoprinosine concentrations up to 800 μg / ml slightly enhanced the antiviral effect of 50 μg / ml isoprinosine.as shows table(1) In vitro experiments have shown that isoprinosine even more strongly reduces infectious adenovirus titers in the presence of interferon-α (IFN-α). Simultaneous the addition of 100 IU / mL and 1000 IU / mL IFN-α and isoprinosine to A549 infected cells with resulted reduction of IC₅₀ values ​​(media concentration of the inhibitor that inhibits infectious titers by 50%) from 9051μg / mL to 8931 μg / mL for HPIV-2 respectively, 9873.75 μg / mL to 7816.5 μg / mL and HAdV-2, respectively as show table (2).

Table 1: Reduction of infectious titers of parainfluenza viruses and adenoviruses examined under the influence of different doses of Isoprinosine  in vitro culture of A549 cells.

Table 2: In vitro antiviral activity of isoprinosine with interferon alpha, during expressed as IC₅₀

Discussion

Isoprinosine has antiviral activity, it also has confirmed immunomodulatory effect.⁽¹⁴⁾ Its effectiveness has been described in randomized and double-blind clinical trials. Deroń analyzing the results Ginsberg et al.⁽¹⁵⁾ experiments confirm good drug tolerance in the macroorganism. After reaching a high concentration in tissues, isoprinosine is metabolized to uric acid and completely eliminated by the kidneys.⁽¹⁶⁾ The study showed that that Isoprinosine is not cytotoxic to A549 cells. isoprinosine at a dose of 800 μg / ml, it also does not change the morphology and does not affect biological activity (in the MTT test) HEp-2 and HEL 299 cell lines.⁽¹⁷⁾ Assessing the effect of isoprinosine on the replication of parainfluenza, and adenoviruses have been shown to isoprinosine after 48 hours. Adenoviruses (HAdV-2) have also been shown to be of the highest susceptibility on the antiviral effect of Isoprinosine among all used in the study of virus strains. In vitro experiments have shown that isoprinosine even more strongly reduces infectious adenovirus titers in the presence of interferon-α (IFN-α). Simultaneous the addition of 100 IU / mL and 100 IU / mL IFN-α and isoprinosine to A549 infected cells with resulted reduction of IC₅₀ values ​​(media concentration of the inhibitor that inhibits infectious titers by 50%) from 9051μg / mL to 8931 μg / mL for HPIV-2 respectively, 9873.75 μg / mL to 7816.5 μg / mL and HAdV-2, respectively. Enhancement of the action of Isoprinosine in the presence of INF-α has been demonstrated also towards the reference strain (Human Herpesvirus 1)HHV-1McIntyre strain.⁽¹⁸⁾ It also turns out to be simultaneous administration of IFN-α and isoprinosine results in improvement of the neurological condition of patients with subacute sclerosing encephalitis (a complication after mumps infection) and helps conventional treatments for local human infections papillomavirus (HPV).⁽¹⁹⁾ Due to the limited ability to control many viral infections it appears reasonable implementation of subsequent experiments to confirm the effectiveness of isoprinosine in inhibiting the replication of clinically or epidemiologically important viruses pathogenic to humans.

Parainfluenza viruses are responsible for respiratory infections that occur with varying severity, depending on the type of virus and the immune function host. On the other hand, adenovirus cause infections in people with various pictures clinical, ranging from upper respiratory tract infections to myositis cardiac or central nervous system infections.⁽²⁰⁾ In this study, IFN-α and isoprinosine have been shown to be added jointly to the virus infected A549 culture RNA reduced adenovirus and parainfluenza virus 2 infectious. Both drugs have documented effects therapeutic also against other viruses.^{(3, 5, 6, 10, 21)} Isoprinosine has been shown at therapeutic doses has no toxic effect; good also has been confirmed drug tolerance.⁽²²⁾ Certainly, the number of publications on biological activity isoprinosine and pharmacokinetic properties of the drug is limited; studies done at various centers confirm that isoprinosine enhances the cellular immune response in cell culture in vivo. Affects the maturation and activation of lymphocytes, increases production of cytokines and stimulates phagocytosis and chemo taxis.⁽²³⁾ isoprinosine is also characterized by pleiotropic antiviral activity, which is, however, secondary to its immunomodulatory properties.⁽²⁴⁾ Linhares et al.2003 ⁽²⁵⁾ showed in vitro experiments that isoprinosine is a synthesis inhibitor Rotavirus RNA. The beneficial effects of isoprinosine in controlling were also highlighted acute viral encephalitis, skin and mucous membrane infections in infected persons herpes simplex viruses, as well as hepatitis A.⁽²⁶⁾ Ochocka et al.2006 ⁽²⁷⁾ describe good isoprinosine tolerance and reduction of illness time due to human alpha herpesvirus infection in children with acute lymphoblastic leukemia treated isoprinosine. Positive pharmacological effect of isoprinosine has also been demonstrated in the treatment of symptomatic human papillomavirus infection in women.⁽²⁸⁾ In a study conducted by Rhoades et al.,⁽²⁹⁾ isoprinosine was shown to influence the dynamics of HIV infection; reduces the level of reverse traxriptase and reduces expression p24 and gp120 on the surface of HIV infected lymphocytes. In addition, it was observed that viability, as well as the number of CD4 + cells and the ratio CD4 + / CD8 + are higher in culture cell treated with isoprinosine compared to HIV infected and non-exposed  isoprinosine cells.^(4,30) In vitro studies have been observed antiviral activity of isoprinosine and inhibiting the multiplication of viruses as a result of the combined administration of isoprinosine and interferon-α . Isoprinosine and IFN-α have been shown to reduce infectious titers of DNA viruses such as human adenoviruses and herpes viruses common.⁽³¹⁾ In patients with sub-acute sclerosing encephalitis (SSPE), associated with measles virus infection, combination therapy is recommended isoprinosine with interferon, due to their likely synergistic actions.⁽³²⁾ Currently, there is no possibility of fully effective causal treatment of infections caused by viruses used in the RNA study. It is also not available specific prevention. Therefore, when confirmed in vivo, demonstrated in an antiviral study of co-administered interferon and isoprinosine, it is possible to include such a combination in controlling infections viral.⁽³³⁾

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