Table 1

Absent toxicity of combinations of compounds in Huh7.5.1 cells and primary human hepatocytes

Compound 1	Concentration	Compound 2	Concentration	Relative Huh7.5.1 viability (%)	Relative PHH viability (%)
(A) Cytotoxic effects of combination therapies on Huh7.5.1 cells and PHH after 48 h
IFN-α2a	10 IU/mL	Anti-CLDN1	10 µg/mL	106±11	101±2
IFN-α2b	10 IU/mL	Anti-CLDN1	10 µg/mL	108±15	97±3
Telaprevir	10 µM	Erlotinib	10 µM	84±21	93±5
Boceprevir	10 µM	Anti-CLDN1	10 µg/mL	94±8	103±3
Simeprevir	10 µM	Anti-CLDN1	10 µg/mL	104±5	97±4
Danoprevir	10 µM	Anti-CLDN1	10 µg/mL	91±12	103±1
Daclatasvir	10 nM	Anti-SR-BI	10 µg/mL	114±18	101±2
Mericitabine	10 µM	Anti-CD81	10 µg/mL	131±22	106±5
Sofosbuvir	10 µM	Anti-SR-BI	10 µg/mL	95±15	110±7
Sofosbuvir	10 µM	Anti-CLDN1	10 µg/mL	93±8	97±5
Sofosbuvir	10 µM	Dasatinib	10 µM	89±14	103±8
Sofosbuvir	10 µM	Erlotinib	10 µM	87±15	101±4
Anti-Fas	10 µg/mL				16±2

Compound 1	Concentration	Compound 2	Concentration	Relative Huh7.5.1 viability (%)
(B) Cytotoxic effects of combination therapies on Huh7.5.1 cells after 5 days
Telaprevir	10 µM	Anti-CLDN1	10 µg/mL	92±4
Telaprevir	10 µM	Erlotinib	10 µM	89±3
Daclatasvir	10 nM	Anti-SB-BI	10 µg/mL	103±16
Daclatasvir	10 nM	Anti-CLDN1	10 µg/mL	106±7
Sofosbuvir	10 µM	Anti-CLDN1	10 µg/mL	93±2
Sofosbuvir	10 µM	Erlotinib	10 µM	91±4
Alisporivir	10 µM	Anti-SR-BI	10 µg/mL	97±8
Alisporivir	10 µM	Anti-CLDN1	10 µg/mL	108±6
Flavopiridol	10 µM			20±6

Compound	CC₅₀
(C) 50% cytotoxic concentrations (CC₅₀) of entry inhibitors in Huh7.5.1 cells. Dose-dependent toxicity of entry inhibitors was measured using Prestoblue assay as described in Materials and methods. CC₅₀ were calculated by regression analysis
Anti-CLDN1*	>1 mg/mL
Anti-CD81*	>1 mg/mL
Anti-SR-BI*	789±189 µg/mL
Erlotinib†	>100 µM
Dasatinib†	>100 µM

Combination	Relative Huh7.5.1 viability (%)
(D) Cytotoxic effects of combination therapies as described in figure 8. Cell viability was measured at the end of the experiment using Prestoblue assay as described in Materials and methods
Telaprevir+mock	97±10
Telaprevir+anti-CLDN1	91±3
Telaprevir+erlotinib	81±4

Cytotoxic effects on Huh7.5.1 cells (A, B, C, D) and PHH (A) using the highest concentrations of each compound used in combination (IFN-α, 10 IU/mL; DAAs, 10 nM or µM; alisporivir, 10 µM; receptor-specific mAbs, 10 µg/mL and PKIs, 10 µM) were assessed by analysing the ability to metabolise MTT after 48 h (A, B), 5 days (C), or 20 days (D) as described.23–32 Anti-Fas antibody (10 µg/mL) or flavopiridol (10 µM) was used as a positive control of toxicity. Toxicity analyses of the most efficient combinations are shown. Data are presented as relative cell viability compared with PHH or Huh7.5.1 cells cultured in the absence of compounds or solvent (=100%). Means±SD from one representative experiment performed in triplicate are shown.
*The stock concentration of the entry inhibitors was 2 mg/mL.
†Erlotinib or dasatinib started to precipitate in the medium at the concentrations higher than 100 µM.
DAA, direct-acting antiviral; IFN, interferon; PHH, primary human hepatocyte; PKI, protein kinase inhibitor; SR-BI, scavenger receptor class B type I.