Antiviral Therapy 2016; 21:535–539 (doi: 10.3851/IMP3028)
Short communication Artesunate demonstrates in vitro synergism with several antiviral agents against human cytomegalovirus Emilien Drouot1, Jocelyne Piret1, Guy Boivin1* Research Center in Infectious Diseases, CHU de Québec – Laval University, Quebec City, QC, Canada
1
*Corresponding author e-mail: [email protected]
Background: Human cytomegalovirus (HCMV) infections remain a major problem in immunocompromised patients. Three antiviral agents, ganciclovir (GCV), foscarnet (FOS) and cidofovir (CDV), are currently approved for the treatment of HCMV infections. They all target the viral DNA polymerase and are associated with significant side effects. Combinations of novel antiviral compounds acting on different targets such as artesunate (ART) with currently approved drugs or eventually letermovir or maribavir (MBV) may result in synergistic effects. Here, we evaluated the in vitro activity of a series of two-drug combinations against a wild-type recombinant HCMV strain by the Gaussia luciferase (GLuc) reporter assay. Methods: The in vitro activity of each drug was first tested individually against HCMV by using the GLuc reporter assay. The activity of two-drug combinations consisting of ART and currently approved drugs,
as well as letermovir or MBV, was then analysed by the Chou-Talalay method. Results: The concentrations of GCV, FOS, CDV and ART that reduced the GLuc activity by 50% (EC50 values) were 3.92 ±1.64 µM, 62.45 ±8.39 µM, 0.68 ±0.19 µM and 3.86 ±1.25 µM, respectively, whereas those of MBV and letermovir were 64 ±22 nM and 2.50 ±0.83 nM, respectively. The combination of ART with GCV, CDV or MBV was associated with synergism, whereas combination of ART with FOS or letermovir resulted in moderate synergism. As expected, the combination of MBV with GCV was antagonistic. Conclusions: These results suggest that the combination of ART with the antiviral agents tested in this study could be an interesting strategy for the treatment of HCMV infections to reduce toxicity and drug-resistance development.
Introduction The morbidity and mortality associated with human cytomegalovirus (HCMV) infections in immunocompromised patients remain important issues despite existing prevention and treatment strategies [1,2]. All currently approved antiviral agents (ganciclovir [GCV], foscarnet [FOS] and cidofovir [CDV]) have the same target, the viral DNA polymerase (pUL54) [3]. The main concerns regarding these antivirals are related to their side effects (that is, nephrotoxicity and myelosuppression) and the emergence of drug-resistant viruses. Moreover, due to common modes of action, cross-resistance to all drugs could occur and leaves clinicians with limited therapeutic options [3]. Several compounds with activity against HCMV that act on novel targets have been described and some of them are currently being evaluated in clinical trials. Letermovir, a new antiviral agent under clinical development, has a ©2016 International Medical Press 1359-6535 (print) 2040-2058 (online)
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novel mode of action by targeting the viral terminase subunit pUL56 and interfering with DNA maturation and packaging [4]. The antiviral drug maribavir (MBV) is a competitive inhibitor of the viral phosphotransferase pUL97 [5] but its clinical development is still uncertain. Artesunate (ART), which is commonly used in the treatment of severe malaria, also demonstrated potent in vitro activity against both susceptible and drug-resistant HCMV isolates [6,7]. ART could interfere with cellular regulatory pathways, which are essential for HCMV replication [8]. Combinations of antiviral agents affecting different viral targets has become a standard strategy for treating some viral infections [9,10]. In this report, we evaluated the in vitro effects of ART combined with currently approved viral DNA polymerase inhibitors and with the new agents letermovir and MBV against HCMV. 535
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Methods The in vitro activity of the different antiviral compounds alone or in combination was evaluated using a recombinant wild-type HCMV strain in human lung fibroblasts (MRC-5; ATCC® CCL-171™, American Type Culture Collection, Manassas, VA, USA) by the Gaussia luciferase (GLuc) reporter assay [11]. The recombinant HCMV was generated from the genome of the reference strain AD169 cloned into a bacterial artificial chromosome in which we integrated the GLuc reporter gene [11]. Newly confluent MRC-5 cells were infected with the recombinant HCMV strain at a multiplicity of infection (MOI) of 0.002. After 90 min, serial twofold dilutions of each antiviral compound in MEM + 2% FBS were added to triplicate wells and the supernatant was then collected on day 6 post-infection to measure the GLuc activity [12]. The concentration of each antiviral compound that reduced the GLuc activity by 50% (EC50) was determined as previously described [12]. Results were reported as the means ± standard deviations of at least six independent experiments for each drug. For two-drug combination studies, the EC50 value of each drug was used to determine an equipotent ratio between the two compounds (that is, EC50-drug1/EC50drug2) as previously described [13]. Serial twofold dilutions of a mixture of the two antiviral compounds at concentrations equivalent to tenfold the EC50-drug1/ EC50-drug2 ratio were added to triplicate wells of cells infected with HCMV at an MOI of 0.002. The GLuc activity was then measured in the supernatant on day 6 post-infection [12]. The Chou–Talalay method based on the median-effect equation derived from the massaction law [13] computed in the CalcuSyn software version 1.0 (Biosoft, Cambridge, United Kingdom) was used to analyse drug interactions for all tested combinations. Briefly, the software extrapolated a combination index (CI) representing the interaction between the
two drugs at specified levels of virus inhibition (that is, 50%, 75%, 90% and 95% as shown in Table 1) from the percentages of GLuc inhibition induced by each drug alone and in combination. A weighted average CI (CIwt) value was then calculated for each combination as (CI50+2×CI75+3×CI90+4×CI95)/10 to assess the drug combinatory effects at high levels of virus inhibition in order to increase therapeutic relevance [13]. Drug combinatory effects were defined as synergism for CIwt 1.45. The viability of MRC-5 cells exposed to ART alone or combined with each antiviral agent was evaluated by real-time cell analysis (RTCA) to evaluate drug-induced cell toxicity. This method measures the variations of impedance using gold microelectrodes integrated in a special culture plate. The cell index, which is derived from the electrical impedance and reflects the density, morphology and adhesion of cells, is measured in realtime for several days. The use of the RTCA system (xCELLigence, ACEA Biosciences, Inc., San Diego, CA, USA) has been previously validated in the context of cytotoxicity studies [14]. Newly confluent MRC-5 cells were exposed to serial twofold dilutions of ART (starting at 640 µM) in the absence or presence of the highest concentration of other antiviral agents tested in susceptibility studies (that is, 40 µM, 600 µM, 5 µM, 20 nM and 2.5 µM for GCV, FOS, CDV, letermovir and MBV, respectively). Cell index values were recorded every 30 min for 6 days and the area under the curve (AUC) was calculated over the 6-day exposure period by using the RTCA Software 2.0. The AUC values were plotted against the logarithm of ART concentrations. The concentration of ART alone or in the different combinations that reduced by 50% the AUC values compared to control cells without drug (CC50) was then determined as previously described [14].
Table 1. Analysis of in vitro effects of two-drug combinations against a wild-type recombinant HCMV strain Drug combination (EC50 ratioa)
Number of independent CI values extrapolated at % of virus inhibitionb Drug combinatory experiments 50 75 90 95 CIwtc effectd
ART + GCV (1:1) 3 ART + FOS (1:15) 3 ART + CDV (8:1) 4 ART + letermovir (2,000:1) 4 ART + MBV (40:1) 3 MBV + GCV (1:40) 4
0.78 ±0.24 0.69 ±0.24 0.67 ±0.31 0.67 ±0.37 0.69 Synergism 0.84 ±0.21 0.76 ±0.08 0.71 ±0.06 0.69 ±0.16 0.72 Moderate synergism 1.34 ±0.24 0.64 ±0.18 0.32 ±0.14 0.21 ±0.11 0.44 Synergism 1.25 ±0.23 0.90 ±0.15 0.76 ±0.17 0.72 ±0.28 0.82 Moderate synergism 1.38 ±0.71 0.67 ±0.47 0.41 ±0.36 0.31 ±0.30 0.52 Synergism 2.35 ±0.67 2.47 ±0.54 2.63 ±0.60 2.77 ±0.78 2.62 Antagonism
a Ratio of EC50 values (concentrations that reduce Gaussia luciferase activity by 50%) of each drug alone (that is, EC50-drug1/EC50-drug2). bCombination index (CI) values extrapolated at indicated % of virus inhibition by use of the CompuSyn Software. CI values represent the means ± standard deviations of three to four independent experiments as indicated. cCIwt, weighted average CI values were calculated as (CI50+2×CI75+3×CI90+4×CI95)/10. dDrug combinatory effects were defined as CIwt 0.7 and 0.9 and 1.2 and 1.45, antagonism. ART, artesunate; CDV, cidofovir; FOS, foscarnet; GCV, ganciclovir; MBV, maribavir.
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Results The EC50 values of the wild-type recombinant HCMV strain to individual antiviral compounds were 3.92 ±1.64 µM, 62.45 ±8.39 µM and 0.68 ±0.19 µM for GCV, FOS and CDV, respectively. As demonstrated previously [15], ART had a potent in vitro activity against HCMV with an EC50 value of 3.86 ±1.25 µM, which was similar to that of GCV. The MBV EC50 value was 64 ±22 nM, which was approximately 65-fold lower than that of GCV [5]. Letermovir was the most potent antiviral agent with an EC50 value of 2.50 ±0.83 nM [16]. The in vitro effect of a series of two-drug combinations consisting of ART with currently approved antiviral drugs or new compounds letermovir or MBV was then evaluated. Figure 1 shows typical CI plots for the two-drug combinations tested with experimental CI values and curves extrapolated at different levels of viral inhibition by the CalcuSyn software. Table 1 shows the
analysis of the effects of different two-drug combinations against the wild-type recombinant HCMV strain by the Chou–Talalay method. The combinations of ART with GCV, CDV or MBV resulted in a synergistic effect (CIwt values of 0.69, 0.44 and 0.52, respectively) whereas the combinations of ART with FOS or letermovir demonstrated a moderate synergism (CIwt value of 0.72 and 0.82, respectively). Finally, MBV combined with GCV resulted in a clear antagonistic effect with a CIwt value of 2.62 as previously reported and in agreement with their competing mechanisms of action [17]. The toxicity of ART alone or in combination with other drugs was next determined in MRC-5 cells. The CC50 value of ART alone was 85.49 ±28.06 µM whereas the values were 91.71 ±16.71 µM, 87.56 ±16.51 µM, 87.21 ±13.16 µM, 77.64 ±7.67 µM and 80.21 ±5.74 µM when ART was combined with GCV, FOS, CDV, letermovir and MBV, respectively. The differences in CC50 values obtained for the two-drug
Figure 1. Representative combination index plots
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Representative combination index (CI) plots for artesunate (ART) with (A) ganciclovir (GCV), (B) foscarnet (FOS), (C) cidofovir (CDV), (D) letermovir and (E) maribavir (MBV) as well as for (F) MBV with GCV at a fixed molar ratio of 1:1, 1:15, 8:1, 2,000:1, 40:1 and 1:40, respectively. Each experimental CI value was plotted against the fraction affected (fa), which represents the inhibitory effect of the two-drug combinations against a wild-type recombinant human cytomegalovirus strain. The curve extrapolated by the CompuSyn software represents the combinatory effect between the two drugs against the viral inhibition levels. The horizontal line drawn at CI=1 corresponds to an additive effect whereas a CI1 indicates synergism and antagonism, respectively. Results are the means ± standard deviations of triplicate wells with two separate measurements and are representative of three to four independent experiments. Antiviral Therapy 21.6
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We should thus expect similar drug combinatory effects when using other ratios of ART combinations tested in our study. This synergistic effect could be explained by the fact that ART may reduce the synthesis of viral proteins through a downregulation of nuclear factor kB (NF-kB) and specificity protein 1 (Sp1) expression, whereas antiviral agents act on their specific viral target to decrease either viral DNA synthesis (GCV, FOS and CDV), encapsidation of viral DNA (letermovir and MBV) or nuclear egress (MBV; Figure 2). In this regard, the combination of brincidofovir, the lipid conjugate of CDV, and MBV that act on different viral targets was also shown to exert a synergistic effect against HCMV in vitro [20]. A recent study indicated that ART alone may be useful for the treatment of mild HCMV diseases due to multidrug-resistant strains but may not be effective against severe HCMV diseases [21]. Treatment of patients with malaria using ART has not been associated with significant adverse effects. The use of ART combined with other antiviral agents could thus be an interesting strategy for the treatment or prevention of infections caused by drug-resistant HCMV viruses by acting on different cellular and viral targets. Combination of ART with GCV could be an alternative treatment of HCMV diseases caused by drug-resistant strains in renal transplant recipients, considering the nephrotoxicity associated with the other approved antiviral compounds. Moreover, the synergistic effect suggested in our study between ART and FOS or CDV
combinations and ART alone were not statistically significant (P>0.05; as assessed by a one-way analysis of variance with Dunnett’s post-test ([n=3 independent experiments]). Thus, the combinatory effects of ART with antiviral agents were mainly due to antiviral cooperation and not to increased cellular toxicity.
Discussion In the present study, we demonstrated a synergistic effect between ART and currently approved antivirals or drugs under clinical development. It has been previously reported that ART combined with GCV demonstrated a strong synergism against HCMV when analysed by another method, that is, the Bliss independency model [18,19], which confirms our results. Other studies also tested combinations of ART with FOS, CDV or MBV [7,15]. However, the latter evaluated only one concentration of both drugs around their respective EC50 values without adequate analysis of the drug combinatory effects. The Chou–Talalay method used in our study demonstrated a synergistic effect for ART and the other tested drugs. Previous studies performed in our laboratory with other antiHCMV drug combinations (for example, letermovir with GCV, FOS and CDV) showed that the use of ratios markedly different from the suggested EC50drug1 to EC50-drug2 ratio did not affect the resulting drug combinatory effect obtained when analysing the data with the CompuSyn software (data not shown).
Figure 2. Proposed model of the synergistic effect of ART with the other antiviral compounds tested in this study
HCMV-infected cell Viral proteins Host proteins NF-κB ART
Sp1
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DNA polymerase (pUL54)
GCV, FOS, CDV
Phosphotransferase (pUL97)
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During the replication process, human cytomegalovirus (HCMV) interferes with cellular pathways to promote the synthesis of viral proteins, especially by up-regulating transcription factors such as nuclear factor kB (NF-kB) and specificity protein 1 (Sp1). The mechanism of action of artesunate (ART) is still unclear but it may reduce the expression levels of NF-kB and Sp1 and thereby decrease the synthesis of viral proteins. By contrast, antiviral agents act directly on viral targets by inhibiting either the viral DNA synthesis (ganciclovir [GCV], foscarnet [FOS] and cidofovir [CDV]), encapsidation of viral DNA (letermovir and maribavir [MBV]) or nuclear egress (MBV). 538
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could improve the efficacy of lower doses of antiviral, which are associated with reduced toxicity, in case of strains with high-level resistance to GCV. In conclusion, our study indicates synergism between ART and several antiviral agents against HCMV. Clinical trials are warranted to confirm these in vitro results.
8. 9. 10. 11.
Acknowledgements This study was supported by a grant from the Canadian Institutes of Health Research (grant MOP-86583 to GB). GB is the holder of the Canada research chair on emerging viruses and antiviral resistance.
Disclosure statement
12.
13. 14.
The authors declare no conflicts of interest.
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Accepted 20 January 2016; published online 4 February 2016
Antiviral Therapy 21.6
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Short communication Artesunate demonstrates in vitro synergism with several antiviral agents against human cytomegalovirus Emilien Drouot1, Jocelyne Piret1, Guy Boivin1* Research Center in Infectious Diseases, CHU de Québec – Laval University, Quebec City, QC, Canada
1
*Corresponding author e-mail: [email protected]
Background: Human cytomegalovirus (HCMV) infections remain a major problem in immunocompromised patients. Three antiviral agents, ganciclovir (GCV), foscarnet (FOS) and cidofovir (CDV), are currently approved for the treatment of HCMV infections. They all target the viral DNA polymerase and are associated with significant side effects. Combinations of novel antiviral compounds acting on different targets such as artesunate (ART) with currently approved drugs or eventually letermovir or maribavir (MBV) may result in synergistic effects. Here, we evaluated the in vitro activity of a series of two-drug combinations against a wild-type recombinant HCMV strain by the Gaussia luciferase (GLuc) reporter assay. Methods: The in vitro activity of each drug was first tested individually against HCMV by using the GLuc reporter assay. The activity of two-drug combinations consisting of ART and currently approved drugs,
as well as letermovir or MBV, was then analysed by the Chou-Talalay method. Results: The concentrations of GCV, FOS, CDV and ART that reduced the GLuc activity by 50% (EC50 values) were 3.92 ±1.64 µM, 62.45 ±8.39 µM, 0.68 ±0.19 µM and 3.86 ±1.25 µM, respectively, whereas those of MBV and letermovir were 64 ±22 nM and 2.50 ±0.83 nM, respectively. The combination of ART with GCV, CDV or MBV was associated with synergism, whereas combination of ART with FOS or letermovir resulted in moderate synergism. As expected, the combination of MBV with GCV was antagonistic. Conclusions: These results suggest that the combination of ART with the antiviral agents tested in this study could be an interesting strategy for the treatment of HCMV infections to reduce toxicity and drug-resistance development.
Introduction The morbidity and mortality associated with human cytomegalovirus (HCMV) infections in immunocompromised patients remain important issues despite existing prevention and treatment strategies [1,2]. All currently approved antiviral agents (ganciclovir [GCV], foscarnet [FOS] and cidofovir [CDV]) have the same target, the viral DNA polymerase (pUL54) [3]. The main concerns regarding these antivirals are related to their side effects (that is, nephrotoxicity and myelosuppression) and the emergence of drug-resistant viruses. Moreover, due to common modes of action, cross-resistance to all drugs could occur and leaves clinicians with limited therapeutic options [3]. Several compounds with activity against HCMV that act on novel targets have been described and some of them are currently being evaluated in clinical trials. Letermovir, a new antiviral agent under clinical development, has a ©2016 International Medical Press 1359-6535 (print) 2040-2058 (online)
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novel mode of action by targeting the viral terminase subunit pUL56 and interfering with DNA maturation and packaging [4]. The antiviral drug maribavir (MBV) is a competitive inhibitor of the viral phosphotransferase pUL97 [5] but its clinical development is still uncertain. Artesunate (ART), which is commonly used in the treatment of severe malaria, also demonstrated potent in vitro activity against both susceptible and drug-resistant HCMV isolates [6,7]. ART could interfere with cellular regulatory pathways, which are essential for HCMV replication [8]. Combinations of antiviral agents affecting different viral targets has become a standard strategy for treating some viral infections [9,10]. In this report, we evaluated the in vitro effects of ART combined with currently approved viral DNA polymerase inhibitors and with the new agents letermovir and MBV against HCMV. 535
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E Drouot et al.
Methods The in vitro activity of the different antiviral compounds alone or in combination was evaluated using a recombinant wild-type HCMV strain in human lung fibroblasts (MRC-5; ATCC® CCL-171™, American Type Culture Collection, Manassas, VA, USA) by the Gaussia luciferase (GLuc) reporter assay [11]. The recombinant HCMV was generated from the genome of the reference strain AD169 cloned into a bacterial artificial chromosome in which we integrated the GLuc reporter gene [11]. Newly confluent MRC-5 cells were infected with the recombinant HCMV strain at a multiplicity of infection (MOI) of 0.002. After 90 min, serial twofold dilutions of each antiviral compound in MEM + 2% FBS were added to triplicate wells and the supernatant was then collected on day 6 post-infection to measure the GLuc activity [12]. The concentration of each antiviral compound that reduced the GLuc activity by 50% (EC50) was determined as previously described [12]. Results were reported as the means ± standard deviations of at least six independent experiments for each drug. For two-drug combination studies, the EC50 value of each drug was used to determine an equipotent ratio between the two compounds (that is, EC50-drug1/EC50drug2) as previously described [13]. Serial twofold dilutions of a mixture of the two antiviral compounds at concentrations equivalent to tenfold the EC50-drug1/ EC50-drug2 ratio were added to triplicate wells of cells infected with HCMV at an MOI of 0.002. The GLuc activity was then measured in the supernatant on day 6 post-infection [12]. The Chou–Talalay method based on the median-effect equation derived from the massaction law [13] computed in the CalcuSyn software version 1.0 (Biosoft, Cambridge, United Kingdom) was used to analyse drug interactions for all tested combinations. Briefly, the software extrapolated a combination index (CI) representing the interaction between the
two drugs at specified levels of virus inhibition (that is, 50%, 75%, 90% and 95% as shown in Table 1) from the percentages of GLuc inhibition induced by each drug alone and in combination. A weighted average CI (CIwt) value was then calculated for each combination as (CI50+2×CI75+3×CI90+4×CI95)/10 to assess the drug combinatory effects at high levels of virus inhibition in order to increase therapeutic relevance [13]. Drug combinatory effects were defined as synergism for CIwt 1.45. The viability of MRC-5 cells exposed to ART alone or combined with each antiviral agent was evaluated by real-time cell analysis (RTCA) to evaluate drug-induced cell toxicity. This method measures the variations of impedance using gold microelectrodes integrated in a special culture plate. The cell index, which is derived from the electrical impedance and reflects the density, morphology and adhesion of cells, is measured in realtime for several days. The use of the RTCA system (xCELLigence, ACEA Biosciences, Inc., San Diego, CA, USA) has been previously validated in the context of cytotoxicity studies [14]. Newly confluent MRC-5 cells were exposed to serial twofold dilutions of ART (starting at 640 µM) in the absence or presence of the highest concentration of other antiviral agents tested in susceptibility studies (that is, 40 µM, 600 µM, 5 µM, 20 nM and 2.5 µM for GCV, FOS, CDV, letermovir and MBV, respectively). Cell index values were recorded every 30 min for 6 days and the area under the curve (AUC) was calculated over the 6-day exposure period by using the RTCA Software 2.0. The AUC values were plotted against the logarithm of ART concentrations. The concentration of ART alone or in the different combinations that reduced by 50% the AUC values compared to control cells without drug (CC50) was then determined as previously described [14].
Table 1. Analysis of in vitro effects of two-drug combinations against a wild-type recombinant HCMV strain Drug combination (EC50 ratioa)
Number of independent CI values extrapolated at % of virus inhibitionb Drug combinatory experiments 50 75 90 95 CIwtc effectd
ART + GCV (1:1) 3 ART + FOS (1:15) 3 ART + CDV (8:1) 4 ART + letermovir (2,000:1) 4 ART + MBV (40:1) 3 MBV + GCV (1:40) 4
0.78 ±0.24 0.69 ±0.24 0.67 ±0.31 0.67 ±0.37 0.69 Synergism 0.84 ±0.21 0.76 ±0.08 0.71 ±0.06 0.69 ±0.16 0.72 Moderate synergism 1.34 ±0.24 0.64 ±0.18 0.32 ±0.14 0.21 ±0.11 0.44 Synergism 1.25 ±0.23 0.90 ±0.15 0.76 ±0.17 0.72 ±0.28 0.82 Moderate synergism 1.38 ±0.71 0.67 ±0.47 0.41 ±0.36 0.31 ±0.30 0.52 Synergism 2.35 ±0.67 2.47 ±0.54 2.63 ±0.60 2.77 ±0.78 2.62 Antagonism
a Ratio of EC50 values (concentrations that reduce Gaussia luciferase activity by 50%) of each drug alone (that is, EC50-drug1/EC50-drug2). bCombination index (CI) values extrapolated at indicated % of virus inhibition by use of the CompuSyn Software. CI values represent the means ± standard deviations of three to four independent experiments as indicated. cCIwt, weighted average CI values were calculated as (CI50+2×CI75+3×CI90+4×CI95)/10. dDrug combinatory effects were defined as CIwt 0.7 and 0.9 and 1.2 and 1.45, antagonism. ART, artesunate; CDV, cidofovir; FOS, foscarnet; GCV, ganciclovir; MBV, maribavir.
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Combinations of artesunate with antivirals against cytomegalovirus
Results The EC50 values of the wild-type recombinant HCMV strain to individual antiviral compounds were 3.92 ±1.64 µM, 62.45 ±8.39 µM and 0.68 ±0.19 µM for GCV, FOS and CDV, respectively. As demonstrated previously [15], ART had a potent in vitro activity against HCMV with an EC50 value of 3.86 ±1.25 µM, which was similar to that of GCV. The MBV EC50 value was 64 ±22 nM, which was approximately 65-fold lower than that of GCV [5]. Letermovir was the most potent antiviral agent with an EC50 value of 2.50 ±0.83 nM [16]. The in vitro effect of a series of two-drug combinations consisting of ART with currently approved antiviral drugs or new compounds letermovir or MBV was then evaluated. Figure 1 shows typical CI plots for the two-drug combinations tested with experimental CI values and curves extrapolated at different levels of viral inhibition by the CalcuSyn software. Table 1 shows the
analysis of the effects of different two-drug combinations against the wild-type recombinant HCMV strain by the Chou–Talalay method. The combinations of ART with GCV, CDV or MBV resulted in a synergistic effect (CIwt values of 0.69, 0.44 and 0.52, respectively) whereas the combinations of ART with FOS or letermovir demonstrated a moderate synergism (CIwt value of 0.72 and 0.82, respectively). Finally, MBV combined with GCV resulted in a clear antagonistic effect with a CIwt value of 2.62 as previously reported and in agreement with their competing mechanisms of action [17]. The toxicity of ART alone or in combination with other drugs was next determined in MRC-5 cells. The CC50 value of ART alone was 85.49 ±28.06 µM whereas the values were 91.71 ±16.71 µM, 87.56 ±16.51 µM, 87.21 ±13.16 µM, 77.64 ±7.67 µM and 80.21 ±5.74 µM when ART was combined with GCV, FOS, CDV, letermovir and MBV, respectively. The differences in CC50 values obtained for the two-drug
Figure 1. Representative combination index plots
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Representative combination index (CI) plots for artesunate (ART) with (A) ganciclovir (GCV), (B) foscarnet (FOS), (C) cidofovir (CDV), (D) letermovir and (E) maribavir (MBV) as well as for (F) MBV with GCV at a fixed molar ratio of 1:1, 1:15, 8:1, 2,000:1, 40:1 and 1:40, respectively. Each experimental CI value was plotted against the fraction affected (fa), which represents the inhibitory effect of the two-drug combinations against a wild-type recombinant human cytomegalovirus strain. The curve extrapolated by the CompuSyn software represents the combinatory effect between the two drugs against the viral inhibition levels. The horizontal line drawn at CI=1 corresponds to an additive effect whereas a CI1 indicates synergism and antagonism, respectively. Results are the means ± standard deviations of triplicate wells with two separate measurements and are representative of three to four independent experiments. Antiviral Therapy 21.6
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We should thus expect similar drug combinatory effects when using other ratios of ART combinations tested in our study. This synergistic effect could be explained by the fact that ART may reduce the synthesis of viral proteins through a downregulation of nuclear factor kB (NF-kB) and specificity protein 1 (Sp1) expression, whereas antiviral agents act on their specific viral target to decrease either viral DNA synthesis (GCV, FOS and CDV), encapsidation of viral DNA (letermovir and MBV) or nuclear egress (MBV; Figure 2). In this regard, the combination of brincidofovir, the lipid conjugate of CDV, and MBV that act on different viral targets was also shown to exert a synergistic effect against HCMV in vitro [20]. A recent study indicated that ART alone may be useful for the treatment of mild HCMV diseases due to multidrug-resistant strains but may not be effective against severe HCMV diseases [21]. Treatment of patients with malaria using ART has not been associated with significant adverse effects. The use of ART combined with other antiviral agents could thus be an interesting strategy for the treatment or prevention of infections caused by drug-resistant HCMV viruses by acting on different cellular and viral targets. Combination of ART with GCV could be an alternative treatment of HCMV diseases caused by drug-resistant strains in renal transplant recipients, considering the nephrotoxicity associated with the other approved antiviral compounds. Moreover, the synergistic effect suggested in our study between ART and FOS or CDV
combinations and ART alone were not statistically significant (P>0.05; as assessed by a one-way analysis of variance with Dunnett’s post-test ([n=3 independent experiments]). Thus, the combinatory effects of ART with antiviral agents were mainly due to antiviral cooperation and not to increased cellular toxicity.
Discussion In the present study, we demonstrated a synergistic effect between ART and currently approved antivirals or drugs under clinical development. It has been previously reported that ART combined with GCV demonstrated a strong synergism against HCMV when analysed by another method, that is, the Bliss independency model [18,19], which confirms our results. Other studies also tested combinations of ART with FOS, CDV or MBV [7,15]. However, the latter evaluated only one concentration of both drugs around their respective EC50 values without adequate analysis of the drug combinatory effects. The Chou–Talalay method used in our study demonstrated a synergistic effect for ART and the other tested drugs. Previous studies performed in our laboratory with other antiHCMV drug combinations (for example, letermovir with GCV, FOS and CDV) showed that the use of ratios markedly different from the suggested EC50drug1 to EC50-drug2 ratio did not affect the resulting drug combinatory effect obtained when analysing the data with the CompuSyn software (data not shown).
Figure 2. Proposed model of the synergistic effect of ART with the other antiviral compounds tested in this study
HCMV-infected cell Viral proteins Host proteins NF-κB ART
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During the replication process, human cytomegalovirus (HCMV) interferes with cellular pathways to promote the synthesis of viral proteins, especially by up-regulating transcription factors such as nuclear factor kB (NF-kB) and specificity protein 1 (Sp1). The mechanism of action of artesunate (ART) is still unclear but it may reduce the expression levels of NF-kB and Sp1 and thereby decrease the synthesis of viral proteins. By contrast, antiviral agents act directly on viral targets by inhibiting either the viral DNA synthesis (ganciclovir [GCV], foscarnet [FOS] and cidofovir [CDV]), encapsidation of viral DNA (letermovir and maribavir [MBV]) or nuclear egress (MBV). 538
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could improve the efficacy of lower doses of antiviral, which are associated with reduced toxicity, in case of strains with high-level resistance to GCV. In conclusion, our study indicates synergism between ART and several antiviral agents against HCMV. Clinical trials are warranted to confirm these in vitro results.
8. 9. 10. 11.
Acknowledgements This study was supported by a grant from the Canadian Institutes of Health Research (grant MOP-86583 to GB). GB is the holder of the Canada research chair on emerging viruses and antiviral resistance.
Disclosure statement
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The authors declare no conflicts of interest.
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Accepted 20 January 2016; published online 4 February 2016
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