Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors

Abstract

Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

FIG. 1.

Dose-dependent inhibition of HCV subgenomic replicon replication in Huh 9-13 cells by Debio 025 (open diamonds), Cs (open squares), VX-950 (closed diamonds), 2′-C-MeCyt (closed triangles), and HCV 796 (closed circles). Huh 9-13 cells were treated for 72 h with various concentrations of the compounds. The levels of HCV RNA were quantified by RT-qPCR and were expressed as the percentage of the level of HCV RNA for the untreated control cells. Data are the mean values of at least three independent experiments.

FIG. 2.

Antiviral effect of the combination of Debio 025 with RBV (A), the protease inhibitor VX-950 (B), the NI 2′-C-MeCyt (C), or the NNI JT-16 (D) in HuH6 cells. The zero plane on the z axis represents an additive effect, the volume above the zero plane indicates synergistic activity, and the volume below the zero plane indicates an antagonistic effect. The different shades represent different ranges of values: black, −20% to 0%; gray, 0% to 20%; dashed line, 20% to 40%. Data for the combinations Debio 025-RBV and Debio 025-2′-C-MeCyt are mean values for three independent experiments; data for the combinations of Debio 025-VX-950 and Debio 025-JT-16 are mean values for two independent experiments.

FIG. 3.

Clearance and rebound experiment with Huh 9-13 cells. (A) Cells were treated for seven consecutive passages with either 1 μg/ml (0.82 μM) Debio 025 (red), 5 μg/ml (7.06 μM) VX-950 (blue), 30 μg/ml (122.85 μM) RBV (green), or 100 IU/ml IFN (black) in the absence of selective G418 pressure. The HCV RNA content was quantified at every passage by means of RT-qPCR and is expressed as the percentage of the HCV RNA content of the untreated (control) sample at the same passage number. (B) During rebound, the compounds were omitted from the culture medium but the cells were again cultured under the selective pressure of G418 (1,000 μg/ml). Following every passage, two rebounds were inserted; on the x axis, the first number refers to the passage number, while the second number stands for the rebound number. Again, the HCV RNA content was quantified and expressed as a percentage of the HCV RNA content of the untreated (control) sample at the same passage and rebound number. (C) Cells were treated for four consecutive passages with 1 μg/ml (0.82 μM) Debio 025, 30 μg/ml (122.85 μM) RBV, and 100 IU/ml IFN in the absence of selective G418 pressure. During rebound, the compounds were omitted from the culture medium but the cells were again cultured under the selective pressure of G418 (1,000 μg/ml). Following every passage, two rebounds were inserted. P, passage; R1, rebound 1; R2, rebound 2.

FIG. 4.

Clearance experiment with Huh 9-13 cells and various concentrations of Debio 025. Cells were treated for seven consecutive passages, in the absence of G418 selective pressure, with the following twofold dilutions of Debio 025: 0.50 μg/ml (0.41 μM) (A), 0.25 μg/ml (0.21 μM) (B), 0.125 μg/ml (0.10 μM) (C), 0.063 μg/ml (0.05 μM) (D), 0.031 μg/ml (0.03 μM) (E), or 0.016 μg/ml (0.01 μM) (F). During rebound, the compounds were omitted from the culture medium but the cells were again cultured under the selective pressure of G418 (1,000 μg/ml). Following every passage, two rebounds were inserted. P, passage; R1, rebound 1; R2, rebound 2. The levels of the remaining HCV RNA were determined by means of RT-qPCR at every passage (and the accompanying rebound passages) and are expressed as a percentage of the amount of total HCV RNA in the untreated (control) cells of the same passage number.

FIG. 5.

Clearance experiment with Huh 9-13 cells and various concentrations of Debio 025 in combination with VX-950. Cells were treated for four consecutive passages with 5 μg/ml (7.06 μM) VX-950 in combination with Debio 025 at either 1 μg/ml (0.82 μM) (open diamonds), 0.5 μg/ml (0.41 μM) (closed squares), 0.25 μg/ml (0.21 μM) (open circles), or 0.125 μg/ml (0.10 μM) (closed triangles) in the absence of G418 selective pressure. The quantity of HCV RNA was measured at every passage by means of RT-qPCR and was normalized against the amount of total RNA extracted from the untreated (control) sample at the same passage number.

FIG. 6.

Colony formation assay. Huh 9-13 cells were treated with Debio 025 alone or in combination with VX-950 (A), BILN 2061 (B), JT-16 (C), or R1479 (D) at the indicated concentrations in the presence of 1,000 μg/ml G418. At the time that the cultures became confluent or a sufficiently large number of colonies had developed, the cells were further passaged under the same experimental conditions. Following 4 weeks of culture, on average, the cells were fixed and stained with Giemsa.