Archives of Medical Research 46 (2015) 8e16

ORIGINAL ARTICLE

In Vitro Evaluation of Synergistic Inhibitory Effects of Neuraminidase Inhibitors and Methylglyoxal Against Influenza Virus Infection Siriwan Charyasriwong,a,* Ken Watanabe,a,* Ratika Rahmasari,a Ayaka Matsunaga,a Takahiro Haruyama,a,b and Nobuyuki Kobayashia,b a

Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan b Central Research Center, AVSS Corporation, Nagasaki, Japan Received for publication July 8, 2014; accepted December 9, 2014 (ARCMED-D-14-0396).

Background and Aims. Influenza virus infections are serious public health concerns worldwide that cause considerable mortality and morbidity. Moreover, the emergence of resistance to anti-influenza viral agents underscores the need to develop new antiinfluenza viral agents and novel treatment strategies. Recently, we identified antiinfluenza viral activity of manuka honey. Therefore, we hypothesized that methylglyoxal (MGO), a key component of manuka honey, may impart anti-influenza viral activity. The aim of this study was to evaluate the anti-influenza viral activity of MGO and its potential in combination treatments with neuraminidase (NA) inhibitors. Methods. MDCK cells were used to evaluate anti-influenza viral activity. To evaluate the mechanism of MGO, plaque inhibition assays were performed. The synergistic effects of MGO and viral NA inhibitors were tested. Results. MGO inhibited influenza virus A/WSN/33 replication 50% inhibitory concentration 5 240  190 mM; 50% cytotoxic concentration 5 1.4  0.4 mM; selective index (SI) 5 5.8, which is related to its virucidal effects. Moreover, we found that MGO showed promising activity against various influenza strains. A synergistic effect was observed by a marked increase in SI of NA inhibitors at |1/100th of their single usage. A synergistic effect of MGO and oseltamivir was also observed against oseltamivirresistant virus. Conclusions. Our results showed that MGO has potent inhibitory activity against influenza viruses and also enhanced the effect of NA inhibitors. Thus, the co-administration of MGO and NA inhibitors should be considered for treatment of influenza virus infections. Ó 2015 IMSS. Published by Elsevier Inc. Key Words: Anti-influenza viral drug, Influenza virus, Manuka honey, Methylglyoxal, Neuraminidase inhibitors, Synergistic effect.

Introduction Influenza viruses are enveloped, negative-stranded RNA viruses with eight segmented genomes belonging to the Orthomyxoviridae family. Two types of the influenza virus, A and B, cause influenza in humans. Influenza A viruses *

These authors contributed equally to this work. Address reprint requests to: Nobuyuki Kobayashi, Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; Phone: þ81 95 819 2456; FAX: þ81 95 819 2898; E-mail: [email protected]

easily mutate, often resulting in the emergence of new antigenic variant subtypes. The threat of a human influenza pandemic has greatly increased over the past 18 years. Highly pathogenic avian influenza viruses, notably the H5N1 virus, emerged in 1997 (1). The 2009 pandemic H1N1 virus quickly spread worldwide (2) and, more recently, human infection with avian influenza H7N9 virus has been reported (3). These outbreaks should serve as warnings to responsible agencies to prepare for the next pandemic threat. At present, two main classes of antiinfluenza viral drugs are available: M2 ion channel inhibitors (amantadine and rimantadine) and neuraminidase

0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.12.002

Anti-influenza Viral Activity of Methylglyoxal

(NA) inhibitors (zanamivir, oseltamivir, laninamivir, and peramivir). The main drawbacks of M2 inhibitors are the rapid development of drug-resistant variants and inefficacy against influenza B virus (4e6). NA inhibitors were developed because of the genetic stability of the NA enzymatic active center among influenza viruses (7). NA has become a promising target for the development of antiviral drugs (8,9). However, influenza viruses have mutated to become resistant to some NA inhibitors, resulting in decreased efficacy of these drugs (10,11). Drug-resistant influenza viruses triggered a serious problem worldwide. For this reason, many researchers are now focused on the development of new anti-influenza treatments (12) or combination therapies to enhance the efficacy of anti-influenza viral drugs (13). Natural products such as microbial metabolites and medicinal plants offer great promise as potentially effective and novel antiviral drugs. To date, several agents isolated from these natural products have been reported. We recently reported that manuka honey, a monofloral honey produced from the nectar of the manuka tree indigenous to New Zealand and Australia, exhibited the highest antiinfluenza viral activity among tested honey samples (14). The a-ketoaldehyde compound methylglyoxal (MGO; molecular weight 72.06; Figure 1A) is present in extremely high concentrations (15) and is the major determinant of the antibacterial activities of manuka honey (16,17). Previous studies indicated that MGO has antiviral activities against foot-and-mouth disease virus (18) and Newcastle disease virus (19). Moreover, our preliminary results showed that the concentration of MGO was 20- to 160fold higher in manuka honey than in other honey samples. Therefore, it is possible that MGO contributes to its antiinfluenza viral activity. The anti-influenza viral activity of MGO was originally reported in 1957 (20) using embryonated chicken eggs. Infection of embryonated chicken eggs is a complicated process and the anti-influenza viral mechanism of action of MGO remains poorly understood. Prior to our report, few attempts have been made to elucidate the anti-influenza virus activity of MGO over the past half century. In this study we investigated the anti-influenza viral activity of MGO and its potential as a combination treatment with NA inhibitors. We found that MGO was effective against various influenza strains, including the 2009 pandemic virus, which is resistant to oseltamivir. In addition, we evaluated the synergistic effect of NA inhibitors and MGO against influenza virus infection.

Materials and Methods Cells, Viruses, and Chemicals MadineDarby canine kidney (MDCK) cells were grown in Eagle’s minimum essential medium (E-MEM) supplemented with 5% fetal bovine serum (FBS) at 37 C in an

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atmosphere of 5% CO2. Influenza virus A strains A/Puerto Rico/8/34 (H1N1), A/Hong Kong/8/68 (H3N2), and A/ duck/Pennsylvania/1/84 (H5N2) were propagated in MDCK cells in the presence of 2.5 mg/mL of trypsin (Sigma-Aldrich Co., St. Louis, MO). The 50% tissue culture infective dose (TCID50) of influenza virus was titrated using MDCK cells. Strains A/WSN/33 (H1N1) and oseltamivir-resistant A/Nagasaki/HA-58/ 2009 (H1N1) (2) were propagated in 10-day-old embryonated chicken eggs for 48 h, after which the infected allantoic fluid was harvested and stored at 80 C until use. Oseltamivir, zanamivir, peramivir, and laninamivir were purchased from F. Hoffmann-La Roche Ltd. (Basel, Switzerland), GlaxoSmithKline PLC (Middlesex, UK), Biocryst, Inc. (Durham, NC), and Daiichi Sankyo, Ltd. (Tokyo, Japan), respectively. Oseltamivir was dissolved in H2O to a concentration of 24 mM, whereas zanamivir and laninamivir were dissolved in DMSO to concentrations of 25 mM and 564.34 mM, respectively. Peramivir (30.5 mM) was directly used without dilution. All samples were maintained at 80 C. Approximately 40% MGO solution (in H2O) was purchased from SigmaAldrich and maintained at 4 C. Prior to performing the experiments, MGO was diluted with E-MEM supplemented with 1% 100  vitamin solution (MEM-vitamin). Evaluation of Anti-influenza Viral and Cytotoxic Activities The anti-influenza viral activity of MGO and NA inhibitors was evaluated as previously described (21) with some modifications. For evaluation of anti-influenza viral activities, MDCK cells were typically seeded in 96-well plates at a density of 3.0  104 cells/well in 100 mL of MEM containing 10% FBS and incubated overnight. Cells were washed with MEM-vitamin. Then, 100 mL of two-fold serially diluted samples (MGO or NA inhibitors in MEMvitamin) were added. Cells were subsequently infected without or with 100 mL of influenza virus A solution (A/ WSN/33, A/Puerto Rico/8/34, A/Hong Kong/8/68, A/ duck/Pennsylvania/1/84, or A/Nagasaki/HA-58/2009 in MEM-vitamin) equivalent to 100 TCID50. The culture plates were incubated at 37 C in an atmosphere of 5% CO2 for 3 days. After incubation of both infected and uninfected cells, the culture medium was removed and 200 mL of 70% ethanol was added for 5 min and then removed. Cells were stained with 200 mL of 0.5% crystal violet (CV) in water for 5 min. After washing with water and air drying, absorbance was measured at 560 nm using an Infinite M200 Tecan plate reader (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The percentage of viable MDCK cells was plotted. To determine the yield of influenza virus, the supernatant from each well was collected and the virus yield was determined using the TCID50 assay. The WST-1 (water-soluble tetrazolium salt) assay and CV staining was used to evaluate the cytotoxicity. Typically,

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Charyasriwong et al./ Archives of Medical Research 46 (2015) 8e16

Figure 1. Anti-influenza viral activity of MGO. Evaluation of the cytotoxicity and anti-influenza viral effect of MGO was performed as described in Material and Methods. (A) Chemical structure of MGO. (B) Cytotoxicity of MGO. MDCK cells grown in 24-well plates were treated with serial dilutions of MGO and left uninfected. Three days after treatment, cytotoxicity of cells was measured by the WST-1 assay (open triangles) or CV staining (closed circles). Relative OD values (%) are expressed as the percentage of cells without MGO treatment. (C) Cell morphology of uninfected MDCK cells treated with increasing concentrations of MGO shown in (B) was compared with those of untreated cells. Bar 5 100 mM. (D) Anti-influenza viral activity of MGO. MDCK cells grown in 24-well plates were treated with (closed symbols) or without (open symbols) 600 TCID50 of A/WSN/33 virus in the presence of MGO. Three days after infection, antiviral activity was measured using the WST-1 assay (triangles) or CV staining (circles). Relative OD values (%) are expressed as the percentage of uninfected cells (open symbols) without MGO treatment. Virus yields in the supernatants were also determined and represented (gray diamonds). (E) Cell morphology of infected MDCK cells treated with increasing concentration of MGO shown in (D) was compared with those of uninfected cells. Bar 5 100 mM. The data are representative of three independent experiments.

Anti-influenza Viral Activity of Methylglyoxal

MDCK cells were seeded in 24-well plates at a density of 1.78  105 cells/well in 1 mL of MEM containing 10% FBS and incubated overnight. Cells were washed with MEM-vitamin and then 1 mL of diluted samples (MGO or NA inhibitors in MEM-vitamin) was added. The culture plates were incubated at 37 C in an atmosphere of 5% CO2 for 3 days. After incubation of both infected and uninfected cells, the medium was replaced with 1 mL of WST-1 reagent at a concentration of 32.6 mg/mL (Dojindo Laboratories, Kumamoto, Japan) in MEM and then incubated at 37 C in an atmosphere of 5% CO2 for 3 h. Absorbance was measured at 450e650 nm using the plate reader. The plates were subsequently fixed and stained with CV and optical density values at 560 nm were determined as described above. Plaque Inhibition Assay The plaque inhibition assay was performed as previously described (14) with some modifications. Approximately 300 plaque-forming units (pfu) of virus in MEM-vitamin were used for infection. The detailed procedures for each treatment are as follows: (i) pretreatment of cells: before plaque inhibitory assays, MDCK cells were pretreated with test samples at 37 C for 1 h. After the medium was removed, cells were washed with MEM and infected by adding the viral suspension containing 300 pfu of virus in MEM-vitamin. (ii) Pretreatment of virus: approximately 107 pfu/mL of virus stock was preincubated with the test samples at room temperature for 1 h. These mixtures were subsequently diluted in MEM-vitamin to obtain |600 pfu/mL and 500 mL aliquots of the diluted mixtures (300 pfu) were used for infection. (iii) The treatment occurred during infection: 250 mL aliquots of the test samples in MEM-vitamin were added to the MDCK cells followed by 250 mL of virus suspension (300 pfu). The cells were then incubated for 1 h. (iv) Treatment of cells after viral infection: after viral infection (300 pfu) for 1 h, the cells were overlaid with 3 mL of agarose solution containing the MGO samples and MEM supplemented with 0.8% agarose, 0.1% bovine serum albumin, and 1% 100  vitamin solution. Evaluation of Synergistic Effects MDCK cells were seeded in 96-well plates at a density of 3.0  104 cells/well in 100 mL of MEM containing 10% FBS and then incubated overnight. NA inhibitors and MGO were serially diluted in each dilution plate in different directions, which is known as the checkerboard method (22). Cells were washed with MEM, after which 100 mL of serially diluted samples (MGO and NA inhibitors) were added, followed by infection with 100 mL of influenza virus solution (A/WSN/33, 100 TCID50; A/ Nagasaki/HA-58/2009, 6.25 and 100 TCID50). The infected culture plates were incubated at 37 C in an atmosphere of 5% CO2 for 3 days. The plates were subsequently fixed and stained with CV and optical density values were determined as described above.

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Statistical Analyses Mean  standard deviations (SD) were calculated from two independent experiments unless otherwise noted; 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) of the samples were calculated using GraphPad Prism software (v.5.01; GraphPad Software, La Jolla, CA) (23). The selective index (SI) was evaluated as the ratio of CC50 to IC50. Results MGO Suppresses Influenza Virus Replication A previous study suggested that several a-ketoaldehyde compounds, including MGO, can suppress influenza virus replication in embryonated chicken eggs (20). However, the precise quantitative evaluation of MGO such as cytotoxicity and anti-influenza viral activity has not yet been fully understood. We first evaluated the cytotoxicity of MGO against MDCK cells using the WST-1 assay and CV staining (Figure 1B). CV staining is an alternative and rapid method for the evaluation of cytotoxicity (24). CC50 values evaluated by the WST-1 assay and CV staining were similar (1.6  0.4 mM vs. 1.4  0.4 mM, respectively). Cell morphology (Figure 1C) seems to be correlated to the relative OD value observed in Figure 1B. We decided to use CV staining for further evaluations. We next evaluated anti-influenza viral activity of MGO and commercial NA inhibitors using MDCK cells (Figure 1D and E and Table 1). The viral cytopathic effect (CPE) was suppressed in the presence of MGO in a dose-dependent manner for all influenza virus strains. IC50 of MGO against A/WSN/33 was 240  190 mM (Figure 1D and Table 1), yielding an SI value (CC50/IC50) of 5.8. In the absence and the presence of 700 mmol MGO, the virus yield was 5.9  105  3.3  105 TCID50/mL and undetectable. The anti-influenza viral activity of MGO against different influenza virus A strains was evaluated and compared to that of commercial NA inhibitors (Table 1). Although NA inhibitors drastically differentially suppressed viral replication depending on the infecting strain, MGO showed only slight differential activity against all strains including an oseltamivir-resistant A/Nagasaki/ HA-58/2009 clinical isolate, which carries the H275Y mutation in the NA gene (2). As expected, A/Nagasaki/HA-58/ 2009 was resistant to oseltamivir (IC50 O 870 mM) and also showed cross-resistance to peramivir (IC50 O 2.5 mM) as reported previously (25). Cell morphology (Figure 1E) seems to be correlated to the relative OD value observed in Figure 1D. These results suggest that MGO suppressed influenza virus replication. MGO Has Virucidal Activity Plaque inhibition assays were performed to determine whether MGO affects influenza virus growth (Figure 2).

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Charyasriwong et al./ Archives of Medical Research 46 (2015) 8e16

Table 1. Efficacy of NA inhibitors and MGO against various strains of influenza virus Compound

CC50a (mM)

MGO

1.4  103  400

Oseltamivir

1.8  103  30

Zanamivir

O100

Laninamivir

O170

Peramivir

O1.5  104

Virus strain WSNc PR8d HKe Duck Penf HA-58g WSN PR8 HK Duck Pen HA-58 WSN PR8 HK Duck Pen HA-58 WSN PR8 HK Duck Pen HA-58 WSN PR8 HK Duck Pen HA-58

Subtype

IC50a (mM)

SIb

H1N1 H1N1 H3N2 H5N2 H1N1 H1N1 H1N1 H3N2 H5N2 H1N1 H1N1 H1N1 H3N2 H5N2 H1N1 H1N1 H1N1 H3N2 H5N2 H1N1 H1N1 H1N1 H3N2 H5N2 H1N1

240  190 360  130 420  140 180  20 250  140 2.5  0.5 9.4  0.9 0.71  0.03 7.9  4.9 O870 0.11  0.02 0.024  0.001 0.11  0.03 1.1  0.02 0.11  0.02 1.2  0.3 1.6  0.1 3.0  0.8 45  4 3.2  0.3 0.011  0.004 0.061  0.020 !0.0050 0.0040  0.0004 O2.5

5.8 3.9 3.3 7.8 5.6 740 190 2.6  103 230 !2.1 O890 O4.1  103 O890 O88 O910 O140 O100 O58 O3.8 O53 O1.4  106 O2.5  105 O3.1  106 O3.5  106 N/A

N/A, not applicable. a IC50: 50% inhibitory concentration, CC50: 50% cytotoxic concentration. b SI: selective index 5 CC50/IC50. c A/WSN/33. d A/Puerto Rico/8/34. e A/Hong Kong/8/68. f A/duck/Pennsylvania/1/84. g A/Nagasaki/HA-58/2009.

For these experiments, MGO was either (i) added to the cells for 1 h and subsequently washed out before viral infection (‘‘pretreatment of cell’’), (ii) mixed with influenza virus solution for 1 h before viral infection (‘‘pretreatment of virus’’), (iii) added during viral adsorption for 1 h and subsequently washed out (‘‘during infection’’) or (iv) added to the agarose gel that overlaid the infected cells (‘‘after infection’’). Pretreatment of cells with 170 mM and 700 mM MGO had slight effects on relative plaque numbers (86.5%  2.6% and 85.7%  0.4%, respectively; Figure 2B). In contrast, plaque formation was completely inhibited when the virus was treated with 170 mM and 700 mM MGO before infection (Figure 2B), suggesting that MGO exhibited potent virucidal activity. In addition, moderate reductions in plaque numbers were obtained by treating cells with these concentrations of MGO during (24.8  2.0% and 2.3  0.1%, respectively; Figure 2B) and after infection (72  10% and 14.7  0.6%, respectively; Figure 2B). As a positive control, the antiinfluenza viral drug zanamivir was added after infection (100 nM) and caused a decrease in plaque numbers (29  3.8%; Figure 2B). Moreover, incubation of influenza virus

with 700 mM MGO for 10 min completely reduced infectivity (Figure 2C). Taken together, these data suggest that MGO has strong virucidal activity. Synergistic Antiviral Effects of MGO in Combination with NA Inhibitors The combined use of antiviral compounds with different mechanisms of action may act synergistically and provide advantages over single-agent treatment, as has been reported in the treatment of human immunodeficiency virus (HIV) (26). NA inhibitors block the release of progeny virions from infected cells, whereas MGO has a virucidal effect on the virus particle prior to infection. The inhibitory effect of NA inhibitors against influenza virus infection markedly increased in the presence of MGO as demonstrated by the decreased IC50 values of NA inhibitors. The IC50 of the NA inhibitors against A/WSN/33 virus when combined with various concentrations of MGO are shown in Table 2. The use of combination treatment tended to reduce the IC50 of NA inhibitors with increasing MGO concentrations. When 170 mM MGO was added together

Anti-influenza Viral Activity of Methylglyoxal

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Figure 2. Virucidal activity of MGO. (A) Plaque formation in the presence of MGO. Confluent monolayers of MDCK cells were grown in 6-well plates and infected with dilutions of virus that produced |300 plaques per well. After 1 h, the virus solution was removed, cells were washed and overlaid with an agarose solution (0.8% agarose in MEM), and plaques were counted after 3 days. For the ‘‘pretreatment of cells’’ experiment, MGO was added to the cells 1 h before infection. For the ‘‘pretreatment of virus’’ experiment, virus and MGO were mixed at room temperature 1 h before addition to the cells. For the ‘‘during infection’’ experiment, MGO/virus solution was added at the beginning of the 1-h infection period. For the ‘‘after infection’’ experiment, MGO was mixed with the agarose solution that was laid over the infected cells. As a control, zanamivir (100 nM) was mixed with agarose solution (virus þ zanamivir). Representative data from duplicate independent experiments are presented. (B) Effect of MGO on plaque numbers. Plaques in Figure 2A were counted and the percentage of plaque inhibition relative to infected controls (virus only) was determined for each drug concentration. Open bar, 170 mM MGO; closed bar, 700 mM MGO; gray bar, without MGO. Means of duplicate samples are shown as relative plaque numbers. Data are presented as the mean  SD. (C) Time-dependent virucidal activity of MGO. Samples were mixed with virus preparations to final MGO concentrations of 170 mM and 700 mM and incubated at room temperature for the indicated time periods. The mixtures were subsequently diluted and plaque assays were immediately performed. Plaque numbers are expressed as a percentage of the number of plaques obtained in the absence of MGO. Data are presented as the mean  SD of duplicate measurements.

with various commercial NA inhibitors, the IC50 decreased to |1/100, 1/300, 1/30, and 1/200th of those values for oseltamivir, zanamivir, laninamivir, and peramivir, respectively. Thus, the SI values of NA inhibitors remarkably increased when the concentration of MGO in the co-treatment increased (Table 3).

Combination Use of MGO with Oseltamivir Against Oseltamivir-resistant Virus Can Improve the Efficacy of Oseltamivir Finally, we tested the synergistic effect of MGO in combination with oseltamivir against oseltamivir-resistant

Table 2. The synergistic effect of combination of MGO and NA inhibitors against A/WSN/33 virus Oseltamivir Concentration of MGO (mM)

IC50 (mM)

0 5.4 22 170

1.8  0.08 2.0  0.31 0.58  0.22 !0.020

a

a

Zanamivir

Laninamivir

Peramivir

Relative ratio

IC50 (mM)

Relative ratio

IC50 (mM)

Relative ratio

IC50 (mM)

Relative ratio

1.0 1.1 0.32 !0.011

0.30  0.19 0.37  0.25 0.046  0.020 !0.0010

1.0 1.2 0.15 !0.0033

0.25  0.01 0.22  0.03 0.14  0.07 !0.010

1.0 0.86 0.53 !0.040

0.028  0.015 0.0055  0.001 0.0033  0.0020 !0.00015

1.0 0.20 0.12 !0.0054

IC50: 50% inhibitory concentration.

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Charyasriwong et al./ Archives of Medical Research 46 (2015) 8e16

Table 3. The combination of MGO and NA inhibitors increases SI value SIa valueb Combinationc

NA inhibitor only

NA inhibitor Oseltamivir Zanamivir Laninamivir Peramivir

740 O890 O140 O1.4  106

9.0 O1.0 O1.7 O1.0

   

104 105 104 108

SI: selective index 5 CC50/IC50. A/WSN/33 was used for evaluation. c In the presence of 170 mM MGO together with each NA inhibitor. a

b

pandemic 2009 H1N1 virus (Table 4). In the absence of MGO, the IC50 value of oseltamivir-resistant A/Nagasaki/ HA-58/2009 was O1000 mM and 200 mM at 100 and 6.25 TCID50/well, respectively. The combination of oseltamivir with increasing MGO concentrations tends to reduce the IC50 value. When 100 mM of MGO was administered with oseltamivir, the IC50 value of oseltamivir decreased to |1/30th at 6.25 TCID50/well. A similar synergistic effect was observed at 100 TCID50/well. In addition, no cytotoxicity of oseltamivir in the presence of 125e500 mM MGO was observed (O1000 mM; data not shown). These results suggest that combined use of MGO with oseltamivir against oseltamivir-resistant virus can improve the efficacy of oseltamivir without affecting cytotoxicity.

Discussion Influenza virus is a serious threat to human health. Thus, there is an urgent requirement for the development of novel anti-influenza viral drugs. In consideration of the findings of a previous report using embryonated chicken eggs (20) and those of our recent report regarding the anti-influenza viral activity of manuka honey (14), we hypothesized that MGO is effective against various influenza viruses, including the pandemic 2009 H1N1 virus as evaluated in Table 4. The synergistic effect of combination of MGO and oseltamivir against oseltamivir-resistant pandemic virus 6.25 TCID50a IC50b

Concentration of MGO (mM) 0 25 100 125 250

100 TCID50

of oseltamivir (mM)

Relative ratio

IC50 of oseltamivir (mM)

Relative ratio

200 110 7.7 !3.9 !3.9

1 0.55 0.038 !0.019 !0.019

O1000 ND ND 448 46

1 N/A N/A !0.45 !0.046

ND, not determined; N/A, not applicable. a TCID50: 50% tissue culture infective dose. b IC50: 50% inhibitory concentration.

the present study using MDCK cells. The presented data indicate that MGO has anti-influenza viral activity (Figure 1 and Table 1), which is most likely due to a virucidal effect, as suggested by the plaque inhibition assay (Figure 2). Furthermore, MGO showed promising activity against multiple influenza virus strains (Table 1) in addition to demonstrating a synergistic effect when administered as a co-treatment with NA inhibitors, as demonstrated by the drastic increase in SI values (Table 3). We found that the IC50 of MGO alone was 180e420 mM (Table 1), which is comparable with values reported in previous studies that demonstrated its inhibitory effects against the proliferation of malaria parasites (IC50 approximately 200 mM) (27) and Escherichia coli and Staphylococcus aureus (IC50 |1.1 mM for each bacterium) (28). Moreover, the antiviral activity of MGO against footand-mouse disease virus (18) and Newcastle disease virus (19) has been reported. Previous reports have shown that a-ketoaldehydes, including MGO, exhibited antiviral activity against influenza viruses (20). They observed that hemagglutination inhibition of the virus occurs in the presence of MGO (2 mM) during an extended incubation period (5 h). We found that a 10-min incubation period in the presence of 700 mM MGO was sufficient to induce virucidal activity (Figure 2C). Our results demonstrated that MGO was effective against various influenza viruses including H1N1, H3N2, H5N2, and oseltamivir-resistant H1N1, suggesting that MGO has a broad spectrum of anti-influenza viral activities, whereas a previous report (20) used the laboratory H1N1 strain A/FM/1/47. Moreover, synergistic antiviral effects of MGO in combination with NA inhibitors were observed when both oseltamivirsensitive and -resistant viruses were used (Tables 2 and 4), thus expanding the findings of previous studies. Although we did not test virucidal activity, it is possible that MGO is also effective against highly pathogenic H5N1, H7N9, and type B viruses. A previous study reported that MGO demonstrated a hemagglutination inhibition effect (20). Thus, MGO may directly interact on the virus surface and interfere with the interaction between viruses and host cells. High rates of oseltamivir resistance were reported in clinical samples worldwide during the 2007e2008 influenza season. In contrast, zanamivir resistance was infrequently observed in clinical isolates (29). Recently, an investigation of possible laninamivir resistance in vitro showed a susceptibility profile similar to that of zanamivir (30). The first emergence of peramivir-resistant clinical isolates was reported during the 2009 pandemic, following prophylaxis or treatment with oseltamivir (29,31). It is generally accepted that two classes of inhibitors that act by different mechanisms exhibit synergistic effects and reduce the rate of drug resistance (32,33). Here, we propose that the mechanism by which MGO exerts anti-influenza viral activity is due to a virucidal effect, whereas NA

Anti-influenza Viral Activity of Methylglyoxal

inhibitors are known to suppress the release of virions from infected cells. Thus, it is reasonable that MGO enhances the efficacy of NA inhibitors. Our result showed that the combined use of NA inhibitors with MGO markedly increased antiviral effect in comparison with that of either drug alone for the laboratory strain A/WSN/33 and the 2009 pandemic strain A/Nagasaki/HA-58/2009. In addition, synergistic combinations can reduce the dose needed to inhibit viral growth (13,34). For example, the absorbed amount of zanamivir and laninamivir administered in oral inhalation dosage form was related to the ability of patients to use this delivery system. An improper technique using this apparatus was shown to be related to low or undetectable serum concentrations of the antivirals (35). For some patients, such as children treated with a combination of MGO and zanamivir or laninamivir, even if insufficient doses of the NA inhibitor were administered, MGO can boost antiinfluenza viral activity and inhibit viral growth and reduce the incidence of drug resistance due to insufficient drug uptake. Moreover, because combination therapies with MGO markedly reduced the IC50 of NA inhibitors, it is possible to reduce the stockpile of neuraminidase inhibitors during future serious influenza pandemics and possibly decrease the emergence of resistant viruses, as is well known in the case of HIV infection. In conclusion, our results showed that MGO has potent inhibitory activity against multiple influenza virus subtypes. In addition, MGO enhanced the effect of currently approved NA inhibitors; therefore, it could be administered to influenza patients together with NA inhibitors. An in vivo study to confirm the efficacy of MGO using mouse infection model is currently in progress. Acknowledgments This work was partly supported by Yamada Research Grant (#0107 and #0131) and a grant from the gCOE Program of Nagasaki University. Conflict of interests: There is no conflict of interest to disclose.

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In vitro evaluation of synergistic inhibitory effects of neuraminidase inhibitors and methylglyoxal against influenza virus infection.

Influenza virus infections are serious public health concerns worldwide that cause considerable mortality and morbidity. Moreover, the emergence of re...
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