Inhibitory Effects of Ambroxol on Influenza A Virus Infection in Vitro

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1 ORIGINAL ARTICLE Takenori Tamaki et al., Inhibitory Effects of Ambroxol on Influenza A Virus Infection in Vitro Takenori Tamaki 1, Hideto Ariumi 1, Hiroaki Kiyohara 2 Kenichi Negishi 3 and Yuji Yoshiyama 1 1 Laboratory of Community Pharmacy, Division of Clinical Pharmacy Research and Education Center for Clinical Pharmacy, School of Pharmacy, Kitasato University, Tokyo, Japan 2 Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan 3 Tokyo University of Science, Faculty of Pharmaceutical Sciences, Chiba, Japan Abstract Mucolytic agents such as L-carbocisteine and ambroxol inhibited influenza-virus proliferation in animal model. L-carbocisteine has been shown to inhibit infections by influenza A virus (IAV) by reducing the expression of its receptor on human tracheal epithelial cells. On the other hand, the inhibitory effects of ambroxol on a replication of IAV are uncertain. To determine the inhibitory effects of ambroxol on IAV infection in MDCK cells, these cells were treated with ambroxol before and after IAV infection. Binding of IAV to sialic acid was not affected when MDCK cells were treated with ambroxol at concentrations of M after IAV infection. However, treating MDCK cells with 125 M ambroxol before the infection markedly reduced the replication of IAV. These results suggested that ambroxol may have inhibited IAV infection by reducing the expression of its receptor on MDCK cells. Key words: Influenza virus, ambroxol, MDCK cell, mucolytic agent, in vitro Introduction The influenza virus is one of the most common infectious pathogens and causes considerable morbidity and mortality, particularly in the aged people, infants, individuals with certain chronic diseases, and immunodeficient patients (Kim et al., 1979, Barker et al., 1980). The clinically used anti-influenza drugs, which include neuraminidase inhibitors such as oseltamivir and zanamivir, and M 2 ion channel blockers such as amantadine, are beneficial for human influenza infection (Hay et al., 1985, Takeda et al., 2002). Amantadine and rimantadine are not so effective due to the frequent emergence of drug-resistant viruses and the lack of inhibitory activity against the influenza B virus (Betakova et al., 1996). The neuraminidase inhibitor oseltamivir is also compromised due to the emergence of drug- resistant viruses that escape from interaction of the inhibitor with the active site of viral neuraminidase (Gubareva et al., 2001). Mucolytic agents, ambroxol (2-amino-3, 5-dibromo-N- [trans-4-hydroxycyclohexyl] benzylamine), has been used for the treatment of chronic bronchitis and neonatal respiratory distress syndrome (Germouty et al., 1987). In additionally, recent study reported that ambroxol significantly suppressed the titer of influenza A virus (IVA) in the airways and improved the survival rate of mice infected with a lethal dose 7

2 of IAV in vivo. It has been clear that ambroxol stimulates the release of suppressors of influenza-virus multiplication, such as pulmonary surfactant (Heath et al., 2006, Kido et al., 1993). In addition, ambroxol transiently suppressed release of the proinflammatory cytokines such as tumour necrosis factor- (TNF- and interleukin-1 (IL-1) into airway fluid (Yang et al., 2002). Recently, mucolytic agent, L-carbocisteine, also was shown to reduce the production of proinflammatory cytokines following infections with rhinovirus (Yasuda et al., 2006). In a replication procedure of IAV, the viruses are attached to SA 2,6Gal, a receptor for human influenza virus on airway epithelial cells and the viruses are then delivered into the cytoplasm, then the RNA of the viruses is released from acidic endosomes into the cytoplasm of the cells. As Yamaya et al. have demonstrated that L-carbocisteine may reduce expression of the receptor in the human airway epithelial cells (Yamaya et al., 2010), whereas the inhibitory effect of ambroxol on a replication of IAV are uncertain. In the present study, we investigate whether ambroxol inhibit influenza A replication in Madin-Darby canine kidney (MDCK) cells. Materials and methods Virus, Cells, and reagents The influenza A virus (H1N1,A/PR/8/34) were purchased from American Type Culture Collection. MDCK cells were cultured as monolayers on MEM medium containing trypsin (2 g/ml) supplemented with 0.42 % heat-inactivated fetal bovine serum (FBS). L-carbocisteine was a generous gift from KYORIN Pharmaceutical Co., Ltd. Ambroxol hydrochloride was purchased from TOKYO CHEMICAL INDUSTRY Co., Ltd.. Oseltamivir phosphate was purchased from CHUGAI PHARMACETICAL Co., Ltd. and used as a positive control. L-carbocisteine and oseltamivir were dissolved in cell culture medium for antiviral evaluations. Ambroxol was initially dissolved in 0.5% dimethyl sulfoxide (DMSO) in MEM. Assessment of cytotoxicity The effects of ambroxol and L-carbocisteine on cell viability were determined using the MTT assay. MDCK cells ( cells/well) were incubated with media in the absence or presence of four-fold diluted ambroxol and L-carbocisteine (final concentration of M) for 3 days. MTT reagents were then added to the monolayer of MDCK cells. After incubation at 37 C for 3 h, absorbance at 570 nm was measured by a spectrophotometer microplate reader. Percentage viability was defined as the relative absorbance of treated cells versus those of untreated control cells. Inhibitory effect of ambroxol on the IAV infection The activities of ambroxol against IAV infection were determined using two different protocols. First, to examine effects of ambroxol on the IAV replication after inoculation with IAV, we measured the release IAV during the first hour after virus infection. In brief, MDCK cells on the culture plate were preincubated with various concentrations of ambroxol and L-carbocisteine in MEM medium for 72 h before the inoculation with IAV for the cell into culture supernatant. After washing the cells with PBS, the cells were inoculated with IAV (10 TCID 50 /ml) for 1 h at room temperature. Cells were washed with EME, and the time course of the release of the IAV was measured using previously described methods (Yamaya et al., 2010). The supernatants were collected at 24 h and 72 h after the inoculation with IAV. As a positive control, MDCK cells were preincubated with oseltamivir before the inoculation with IAV. The viral infectivity titer of supernatant fluids was determined by observing the cytopathogenic effects (CPE) of the influenza virus on the MDCK cells. MDCK cells was cultured with 50 L of each 10-fold serial dilution of the culture supernatant in 96 well microplates for 4 days, and virus-induced cytopathic effects were observed under an inverted microscope. The viral titer (TCID 50 /ml) was calculated using the Reed-Muench method (Reed et al., 1938). In the second protocol, to examine the inhibitory effects of ambroxol on IAV binding to sialic acid in the Hemagglutination inhibition (HAI) assay, HAI assay was carried out as described previously (Song et al., 2005,). MDCK cells were infected with 100 L of IAV (100 8

3 TCID 50 /ml) per well for 1 h at 37 C. After the inoculation with IAV, cells were washed with PBS (ph mM NaCl, 14mM Na 2 HPO 4, 1.5mM KH 2 PO 4 and 2.7mM KCl) and incubated with PBS containing various concentrations of ambroxol and L-carbocisteine for 72 h at 37 C. All supernatants for the virus growth inhibition assay were collected and used for titration of the infectious virus based on the hemagglutination of chicken red blood cells. Briefly, supernatants were serially diluted 4-fold and incubated with an equal volume of a 1 % chicken red blood cell suspension. Viral titers were then calculated as described previously (Al-Jabri et al., 1996). Statistics Results were expressed as the mean ± SEM, and statistical analysis was performed using a one-way measure of ANOVA. Post hoc analysis was performed where appropriate using the Tukey method. Values of p<0.05 were assumed to be significant for all analyses. Results Cellular toxicity of ambroxol and L-carbocisteine We evaluated the cytotoxicities of ambroxol and L-carbocisteine using the MTT assay. MDCK monolayers were incubated with media in the absence or presence of four-fold diluted ambroxol and L-carbocisteine for 72 h. MTT reagents were then added to the MDCK monolayer. After incubation at 37 C for 3 h, absorbance (570 nm) was measured by a spectrophotometer microplate reader. When the dose of ambroxol and L-carbocisteine reduced cell viability by 50 %, it was considered to have affected MDCK cells. The results showed that M of ambroxol and L-carbocisteine did not reduced cell viability by 50 % (data not shown). Effects of ambroxol on IAV infection Evidence for the continuous production of the virus was obtained by demonstrating that each of the supernatant fluids collected between 1-24 h and h after infection contained the influenza virus. The treatment of cells with ambroxol and oseltamivir before injection significantly decreased viral titer levels in supernatant fluids from 1-24 h after infection. However, the inhibitory effects of ambroxol and oseltamivir decrease to viral titer levels in supernatant fluids from h after infection (Fig. 1, Fig. 2). Ambroxol significantly inhibited the replication of influenza A when added 1 h after inoculation with the virus. Inhibitory effects of ambroxol on IAV hemagglutination The IAV has the ability to adsorb to chicken red blood cells (RBC), thereby resulting in hemagglutination (Wiley et al., 1987, Suzuki et al., 1994). We examined the inhibitory effects of ambroxol and L-carbocisteine on IAV binding to sialic acid in the HA assay. As shown in Fig 3, no inhibition on HA titer was observed with the ambroxol and L-carbocisteine treatment. Discussion In the present study, we demonstrated that ambroxol reduced viral titer of IAV in the supernatant of fluids of MDCK. A previous study reported that ambroxol significantly suppressed proliferation of the influenza virus in the airways and improved the survival rate of mice infected with a lethal dose of the IAV in vivo (Yang et al., 2002). For evaluations of the therapeutic effects of medicines, mammalian models are used. However, experiments using mammals, such as mice and rats, are problematic from an ethical perspective of animal welfare. Experiments with model animals should be performed following the 3Rs (Replacement, Reduction, Refinement), replaces animal testing with experiments that use non-animal systems for the purpose of research, education, toxicity testing, or production (Replacement), and includes reductions in the use of animals (Reduction) and lessening or eliminating pain or distress to animals (Refinement). Therefore, we determined whether ambroxol prevented IAV infection in vitro using Madin Darby canine kidney (MDCK) cells. The present result indicated that pretreatment of cells with ambroxol and oseltamivir markedly decreased viral titers of the IAV in supernatant fluids from 24 h after inoculation with the virus (Fig. 1, Fig. 2). In previous studies, Ambroxol has been shown to stimulate the release of suppressor of influenza-virus multiplication, such as pulmonary surfactant (Benne et al., 1995) and 9

4 mucus protease inhibitor (Beppu et al., 1995), in addition that ambroxol suppressed releases of tumour necrosis factor- (TNF- ) and interleukin-1 (IL-1) into airway fluid (Yang et al., 1995). The inflammatory mediators such as TNF- and IL-1 in airways increase the expression of SA 2,6Gal glycotopes on serum glycoproteins (Yasukawa et al., 2005). In an infection of Flu A virus, the viruses are attached to SA 2,6Gal, a receptor for human influenza virus on airway epithelial cell (Rogers et al., 1983). The viruses are then delivered into the cell by an endosome. Then the viral ribonucleic acid (RNA) replicates and undergoes translation, and new virions bud from the surface of the host cell and are released to infect other cells in the host. Fig. 1 Viral release in supernatant fluids of MDCK cells obtained at different times (A: 24 h, B: 72 h) after exposure to tissue culture infective dose (TClD 50 )/cell IAV in the presence of ambroxol or vehicle of ambroxol. Results were means ± SEM from 3 different. Significant differences from viral infection alone are indicated by *P<0.05. Fig.2 Viral release in supernatant fluids of MDCK cells obtained at different times (A: 24 h, B: 72 h) after exposure to tissue culture infective dose (TClD 50 )/cell IAV in the presence of Oseltamivir (10 mg/ml) was used as a positive control or vehicle of Oseltamivir. Results were means ± SEM from 3 different. Significant differences from viral infection alone are indicated by *P<

5 Fig. 3 Receptor binding affinity of (A) ambroxol and (B) L-carbocisteine determined by HA assay. Hemagglutination properties of viruses were determined using red blood cells. In fact, ambroxol transiently suppressed release of the cytokine, TNF- and IL-1 into airway fluid (Yang et al., 2002), L-carbocisteine also was shown to inhibit the viral titers in supernatant fluids following infections with rhinovirus via reducing the production of proinflammatory cytokines (Yasuda et al., 2006). IAV has an ability to adsorb to chicken red blood cells resulting in hemagglutination. The viral spike glycoprotein hemagglutinin (HA) binds to sialic acid on the surface of MDCK cells and mediates membrane fusion for the viral uncoating process. We examined the inhibitory effects of ambroxol and L-carbocisteine on IAV binding to sialic acid using the HAI assay. The hemagglutination of erythrocytes with IAV is mediated by the interaction between HA and a terminal sialic acid linked to sugar chains on the surfaces of erythrocytes. However, ambroxol exhibited no hemagglutinin inhibition activity over that of the control (Fig. 3). The results indicate that ambroxol do not have directly inhibitor of the receptor binding activity. The main objective of the present studies was to test the working hypothesis that ambroxol can reduce viral titers in supernatant fluids in MDCK cell after IAV infection, further studies are needed to clarify how ambroxol can reduce viral titer in vitro for the treatment or prevention of influenza-virus infection. References Al-Jabri A.A., Wigg M.D. and Oxford J.S. (1996) Initial in vitro screening of drug candidates for their potential antiviral activities. In: Kangro H, Mahy B, editors. Virology Methods Manual. London: Academic Press. pp Barker, W.H. and Mullooly, J.P. (1980) Impact of epidemic type A influenza in a defined adult population. Am J Epidemiol, 112, Beppu, Y., Imamura, Y., Tashiro, M., Towatari, T., Ariga, H. and Kido H. (1997) Human mucus protease inhibitor in airway fluids is a potential defensive compound against infection with influenza A and Sendai viruses. J Biochem, 121, Betakova T., Nermut M.V. and Hay AJ. (1996) The NB protein is an integral component of the membrane of influenza B virus. J Gen Virol., 77, Germouty, J. and Jirou-Najou, J. (1987) Clinical efficacy of ambroxol in the treatment of bronchial stasis. Respiration, 51,

6 Gubareva LV, Webster RG, and Hayden FG. (2001) Comparison of the activities of zanamivir, oseltamivir, and RWJ against clinical isolates of influenza virus and neuraminidase inhibitor-resistant variants. Antimicrob Agents Chemother. 45, Hay A.J., Wolstenholme A.J., Skehel JJ and Smith MH. (1985) The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 4, Heath, M.F. and Jacobson, W. (1985) The inhibition of lysosomal phospholipase A from rabbit lung by ambroxol and its consequences for pulmonary surfactant. Lung, 163, de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., Ha, do Q., Guan, Y., Peiris, J.S., Chinh, N.T., Hien, T.T. and Farrar, J. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med, 12, Kido, H., Sakai, K., Kishino, Y. and Tashiro, M. (1993) Pulmonary surfactant is a potential endogenous inhibitor of proteolytic activation of Sendai virus and influenza A virus. FEBS Lett., 322, Kim, H.W., Brandt, C.D., Arrobio, J.O., Murphy, B., Chanock, R.M. and Parrott, R.H. (1979) Influenza A and B virus infection in infants and young children during the years Am J Epidemiol., 109, Reed, L.J. and Muench, H. (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg., 27, Rogers, G.N. and Paulson, J.C. (1983) Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin. Virology, 127, Song, J.M., Lee, K.H. and Seong, B.L. (2005) Antiviral effect of catechins in green tea on influenza virus. Anitiviral Res, 68, Suzuki Y. (1994) Gangliosides as influenza virus receptors. Variation of influenza viruses and their recognition of the receptor sialo-sugar chains. Prog. Lipid Res., 33, Takeda M., Pekosz A., Shuck K., Pinto L. H. and Lamb RA. (2002) Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture. J Virol. 76, Tashiro M., Yokogoshi Y, Tobita K, Seto JT, Rott R, Kido H. (1992) Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopathogenicity. J Virol., 66, Wiley D.C., Skehel J. J. (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem., 56, Yamaya M., Nishimura H., Shinya K., Hatachi Y., Sasaki T., Yasuda H., Yoshida M., Asada M., Fujino N., Suzuki T., Deng X., Kubo H. and Nagatomi R. (2010) Inhibitory effects of carbocisteine on type A seasonal influenza virus infection in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol., 299, L Yang B., Yao D.F., Ohuchi M., Ide M., Yano M., Okumura Y. and Kido H. (2002) Ambroxol suppresses influenza-virus proliferation in the mouse airway by increasing antiviral factor levels. Eur Respir J., 19, Yasuda H., Yamaya M., Sasaki T., Inoue D., Nakayama K., Yamada M., Asada M., Yoshida M., Suzuki T., Nishimura H. and Sasaki H. (2006) Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells. Eur Respir J., 28, Yasukawa Z., Sato C. and Kitajima K. (2005) Inflammation-dependent changes in alpha2,3-, alpha2,6-, and alpha2,8-sialic acid glycotopes on serum glycoproteins in mice. Glycobiology, 15(9), (Received: May 27, 2014/ Accepted: September 25,2014) Corresponding author: Yuji Yoshiyama, Ph.D. Laboratory of Community Pharmacy, Division of Clinical Pharmacy, Research and Education Center for Clinical Pharmacy School of Pharmacy, Kitasato University Shirokane, Minato-ku, Tokyo, , Japan Tel: Fax: yoshiyamay@pharm.kitasato-u.ac.jp 12

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