Interaction of immunosuppressants with HCV antivirals daclatasvir and asunaprevir: combined effects with mycophenolic acid

Figure 1 Hepatitis C virus replication is effectively inhibited by daclatasvir and asunaprevir. Huh7-ETluc cells were cultured with increasing concentrations of DCV (A) or ASV (B). The luciferase activity in these cells is a direct measure of HCV replication. HCV replication was significantly inhibited by 0.01 and 0.1 nmol/L DCV and 1 and 10 nmol/L ASV (mean of 13 independent experiments performed in triplicate, P < 0.001 Mann-Whitney test); The luciferase signal in Huh7-PGK-luc cells, stably expressing luciferase, was not affected by any concentration of DCV (C) or ASV (D), indicating that the observed effect in HUh7-ETluc is not due to non-specific inhibition of luciferase (mean of 7 experiments performed in triplicate); HCV replication in the infectious JFH model was effectively inhibited by DCV (E) at all tested concentrations (mean of 4-6 independent experiments measured in duplicate, P = 0.004 for 0.01 nmol/L DCV, P = 0.11 for 0.05 nmol/L DCV and P = 0.007 for 0.1 nmol/L DCV), as well as by 5 nmol/L and 10 nmol/L ASV (F) (mean of 4-6 independent experiments measured in duplicate, P = 0.01 for 5 nmol/L ASV and P = 0.007 for 10 nmol/L ASV). HCV: Hepatitis C virus; DCV: Daclatasvir; ASV: Asunaprevir; MPA: Mycophenolic acid; DAAs: Direct acting antivirals.

Figure 2 Dose-dependent inhibition of hepatitis C virus replication by daclatasvir and asunaprevir is not affected by rapamycin and tacrolimus. A, B: Huh7-ETluc cells were cultured with increasing concentrations of DCV (A) or ASV (B), in combination with different concentrations of RAPA; C, D: Huh7-ETluc cells were cultured with increasing concentrations of DCV (C) or ASV (D), in combination with different concentrations of TAC. After 24 h incubation luciferase was measured. HCV replication was effectively inhibited by ASV and DCV but not by rapamycin. RAPA and TAC had no effect on the antiviral action of ASV and DCV. Results are mean ± SE of 3 independent experiments performed in triplicate. HCV: Hepatitis C virus; DCV: Daclatasvir; ASV: Asunaprevir; RAPA: Rapamycin; TAC: Tacrolimus.

Figure 3 Calcineurin inhibitor cyclosporine A does not affect the antiviral activity of direct acting antivirals. A, B: Huh7-ETluc cells were cultured with increasing concentrations of DCV (A) or ASV (B), in combination with different concentrations of CSA. After 24 h luciferase was measured. HCV replication was inhibited by CSA: 5 μg/mL CSA significantly inhibited HCV replication compared to control (Mann-Whitney test, P = 0.03 for DCV, P = 0.04 for ASV). The antiviral action of DCV of ASV was not negatively affected by CSA and vice versa; C, D: The luciferase signal in Huh7-PGK-luc cells, stably expressing luciferase, was not affected by any concentration of DCV (C) or ASV (D) with or without CSA, indicating that the observed effect in HUh7-ETluc is not due to non-specific inhibition of luciferase. Results are mean ± SE of 4 independent experiments performed in triplicate; E, F: In the JFH infectious HCV cell culture model, HCV RNA levels were inhibited to by > 99% of control levels by both DCV (E) and ASV (F). The addition of 0.5 μg/mL CSA completely inhibited HCV replication (E, F). Shown are the results of two independent experiments, measured in duplicate by RT-qPCR. CSA: Cyclosporine A; HCV: Hepatitis C virus; DCV: Daclatasvir; ASV: Asunaprevir.

Figure 4 Daclatasvir and asunaprevir show a combined antiviral effect with mycophenolic acid. A, B: Huh7-ETluc cells were cultured with increasing concentrations of DCV (A) or ASV (B), in combination with different concentrations of MPA. After 24 h incubation luciferase was measured. HCV replication was effectively inhibited by ASV and DCV and by increasing concentrations of MPA. As shown in the bar graphs, when cells were treated with 0.1 nmol/L DCV or 10 nmol/L ASV, the addition of 1 and 5 μg/mL MPA further significantly inhibited HCV replication (P = 0.02 for 1 μg/mL and P = 0.08 for 5 μg/mL MPA and 0.1 nmol/L DCV; P = 0.01 for 1 μg/mL, 5 μg/mL MPA and 10 nmol/L ASV, Mann-Whitney test). Results are means ± SEM of 4 or 5 independent experiments performed in triplicate; C, D: The luciferase signal in Huh7-PGK-luc cells, stably expressing luciferase, was not affected by any concentration of DCV (C) or ASV (D) with or without MPA, indicating that the observed effect in HUh7-ETluc is not due to non-specific inhibition of luciferase. Results are mean ± SE of 3 independent experiments performed in triplicate; E: In the Huh7 infectious model, 5 μg/mL MPA reduced HCV replication by 68% of control levels. The inhibition of HCV replication by DCV was further reduced by MPA. The highest dose of DCV (0.1 nmol/L) inhibited HCV replication by 96.5% of control levels with an extra reduction by 99.4% of control levels by MPA; F: When HCV infected Huh7 cells were treated with 10 nmol/L ASV, HCV replication was reduced by 54% of control levels, with no additional effect of MPA. Results are mean of 6 experiments, performed in duplicate. HCV: Hepatitis C virus; DCV: Daclatasvir; ASV: Asunaprevir; MPA: Mycophenolic acid.

Figure 5 The combined antiviral action of DAAs with MPA is not caused by increased expression by ISGs and is partly reversed by guanosine. A: The expression of IRF1, IRF9, and IFITM3 was upregulated after 48h culture with 5 mg/ml MPA. 0.1 nM DCV and 10 nM ASV had no effect on the expression of these genes and did not affect the MPA induced expression. The results are means ± SEM of 2 independent experiments, performed in duplicate; B: The effect of guanosine (GU) supplementation on the combined antiviral action of DCV and ASV with MPA was investigated in Huh7ET-luc cells: MPA inhibited HCV replication by 69% of control, and this was significantly reversed by the addition of 50 μmol/ml guanosine by 30% of control (Mann-Whitney test, P = 0.03). Guanosine did not affect the antiviral action of DSV or ASV, and significantly reversed the combined antiviral effect of DSV or ASV with MPA (Mann-Whitney test P = 0.03 for DSV + MPA and P = 0.03 for ASV + MPA) Results are mean of 4 independent experiments performed in triplicate; C: In the infectious JFH model, HCV replication was effectively inhibited by 5mg/ml MPA, 0.1 nM DCV and 10nM ASV (by 68%, 96.5% and 54% of control respectively).The addition of 50 μmol/ml guanosine partly reversed the antiviral action of MPA by 49% of control, and had no effect on the antiviral action of DSV or ASV, either in the absence or presence of MPA. Results are mean ± SEM of 4-6 independent experiments, performed in duplicate.