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. 2010 Feb;54(2):660-72.
doi: 10.1128/AAC.00660-09. Epub 2009 Nov 23.

SCY-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis C virus RNA replication in vitro

Sam Hopkins  1 , Bernard Scorneaux, Zhuhui Huang, Michael G Murray, Stephen Wring, Craig Smitley, Richard Harris, Frank Erdmann, Gunter Fischer, Yves Ribeill
Affiliations

SCY-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis C virus RNA replication in vitro

Sam Hopkins et al. Antimicrob Agents Chemother. 2010 Feb.

Abstract

SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro. SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 microM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro. SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV.

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Figures

FIG. 1.
FIG. 1.
Chemical structures of CsA (I) and SCY-635 (II). SCY-635 differs from CsA at two positions. The alpha carbon atom of sarcosine at position 3 contains the dimethylamino-ethylthio substituent. The gamma carbon atom of the N-methyl leucine at position 4 contains a hydroxyl group.
FIG. 2.
FIG. 2.
Suppression of HCV replication in vitro. The anti-HCV activity of SCY-635 was assessed by using the HCV subgenomic replicon system. HCV RNA replication was assessed by quantifying HCV replicon-derived luciferase activity. The antiviral activity of SCY-635 was evaluated at 24, 48, 72, and 120 h after drug treatment. Exposure of replicon cells to SCY-635 reduced the level of HCV replication in a time- and a dose-dependent manner.
FIG. 3.
FIG. 3.
Inhibition of PPIase activity of CyPA by CsA and by SCY-635. PPIase activity was determined in a coupled assay with chymotrypsin by using N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide as the substrate. Determinations were performed in duplicate. Inhibition constants were determined by fitting the data to a model for tightly binding reversible enzyme inhibitors.
FIG. 4.
FIG. 4.
Inhibition of calcineurin phosphatase activity by the binary complex formed between CsA and human CyPA and by the binary complex formed between SCY-635 and human CyPA. Calcineurin phosphatase activity was determined by measuring the dephosphorylation of p-nitrophenyl phosphate. CsA or SCY-635 was incubated in a 1:1 molar ratio with CyPA in the presence of calcineurin (0.00132 μM) and calmodulin (0.050 μM) at 22°C for 30 min. All determinations were performed in triplicate. Mean values were expressed as the percentage of calcineurin activity calculated relative to that for an inhibitor-free control.
FIG. 5.
FIG. 5.
SCY-635 is a weaker inhibitor of IL-2 secretion than CsA. Jurkat cells (2 × 105 cells/well) were pretreated for 1 h at 37°C with either SCY-635 (concentration range, 0.0015 μM to 10 μM) or CsA (concentration range, 0.000033 μM to 0.416 μM). Cells were induced with a mixture of anti-CD3 and anti-CD28 antibodies at 0.4 μg/well and 2 μg/well, respectively. Sample supernatants were collected for measurement of the level of IL-2 production at the end of a 6-h incubation.
FIG. 6.
FIG. 6.
SCY-635 exhibits synergistic antiviral activity when it is used in combination with rIFNα-2b. SCY-635 was tested at nine concentrations (range, 0.008 to 2.0 μM) with rIFNα-2b at five concentrations (range, 0.005 to 0.5 μM). After a 72-h incubation period, the cells were processed to determine antiviral activity (luciferase) and the data were analyzed using the Prichard and Shipman MacSynergy II data analysis program.
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