Potential conflict of interest: Dr. Ruegg received grants from Debiopharm.
Cyclosporin A (CsA) inhibits replication of the HCV subgenomic replicon, and this effect is believed to not be mediated by its immunosuppressive action. We found that DEBIO-025, a novel non-immunosuppressive cyclophilin inhibitor derived from CsA, inhibited HCV replication in vitro more potently than CsA. We also examined the inhibitory effect of DEBIO-025 on naive HCV genotypes 1a or 1b in vivo using chimeric mice with human hepatocytes. These mice were treated for 14 days with DEBIO-025, pegylated-interferon α−2a (Peg-IFN), a combination of either drugs, or CsA in combination with Peg-IFN. In mice treated with Peg-IFN, serum HCV RNA levels decreased approximately 10-fold whereas DEBIO-025 treatment alone did not induce any significant change. In mice treated with both DEBIO-025 and Peg-IFN, HCV RNA levels decreased more than 100-fold. All mice treated with Peg-IFN combined with CsA died within 4 days. The combination treatment of DEBIO-025 and Peg-IFN reduced HCV RNA levels and core protein expression in liver, indicating that the HCV RNA levels reduction in serum was attributable to intrahepatic inhibition of HCV replication. Conclusion: We demonstrated that DEBIO-025 was better tolerated than CsA, and that its anti-HCV effect appeared to be synergistic in combination with Peg-IFN in vivo. (HEPATOLOGY 2007;45:921–928.)
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Hepatitis C virus is a small enveloped RNA virus that belongs to the Flaviviridae family.1 A hallmark of HCV infection is its high propensity to establish a persistent infection that evades the host immune response, leading to chronic liver disease, chronic hepatitis, cirrhosis, and hepatocellular carcinoma.2, 3 Although approximately 170 million individuals are infected with HCV worldwide, drugs that are specifically active against hepatitis C are not yet available.
Currently, the main therapy for chronic hepatitis C is a combination of pegylated interferon alpha (Peg-IFN) and ribavirin. In the intention-to-treat analysis, this combination therapy led to a sustained virological response in approximately 55%4, 5 of patients infected with any HCV genotype and in 42%4 to 46%5 of patients with genotype 1. The results of clinical trials were based on selected patients. The proportion of elderly patients was low, and patients with HBV or HIV coinfection, renal disease, post-transplantation status, or hematological disorders were excluded.4–8 Because approximately 50% of patients show a poor response to combined treatment with Peg-IFN and ribavirin, effective therapies are urgently needed.
We previously reported that combination therapy of interferon (IFN) α-2b and cyclosporin A (CsA) for 24 weeks produced a sustained virological response in 42% of patients with both HCV genotype 1b and high viral levels.9 High blood levels of CsA correlate with virological response during treatment for HCV, but occasionally can cause adverse events related to immunosuppression.10 CsA also suppresses HCV replication in vitro, by inhibiting the interaction between HCV nonstructural protein 5B and cyclophilin.11
CsA is an immunosuppressive agent widely used to improve graft survival after organ transplantation.12 It was isolated as a metabolite from Beauveria nivea and consists of a cyclic polypeptide of 11 amino acids.13 DEBIO-025 is a synthetic compound showing a more potent cyclophilin inhibitory activity as compared with CsA14 and differing from CsA by the substitution of 2 amino acids (Fig. 1A; see Materials and Methods).15 DEBIO-025 lacks immunosuppressive effects, although it still has remarkable inhibitory effects on HCV replication in vitro.16
We report the in vivo effectiveness and tolerability of DEBIO-025 administered in combination with Peg-IFN in chimeric mice with human hepatocytes that were infected with HCV genotypes 1a or 1b.
DEBIO-025 is a synthetic compound derived from CsA. Sarcosine (N-methyl-D-glycine) at position 3 and N-methyl-D-leucine at position 4 are substituted for N-methyl-D-alanine and N-ethyl-D-valine, respectively (Fig. 1A).16 DEBIO-025 was obtained from Debiopharm (Lausanne, Switzerland). CsA was purchased from Fluka Chemie (Buchs, Switzerland), and Peg-IFN was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan).
Anti-HCV Assay in HuH-7 Cells Harboring Subgenomic Replicons.
We used 2 HCV subgenomic replicon cell lines, FLR3-117 and R6FLR-N,18 which were constructed as shown in Fig. 2A. They were seeded at a density of 5 × 103 per well in 96-well tissue culture plates, in complete Dulbecco's modified Eagle's medium GlutaMax I (DMEM-GlutaMaxI; Invitrogen, Carlsbad, CA) and containing 5% fetal bovine serum (Invitrogen).17, 18 The genome of the 2 replicons was genotype 1b. After incubation for 24 hours at 37°C (5% CO2), the medium was removed, and serial dilutions of DEBIO-025 or CsA in growth medium were added. After 72 hours, luciferase activity was determined using the Bright-Glo luciferase assay kit (Promega Madison, WI). The luciferase signal was measured in triplicate using an LB940 luminometer (Berthold, Freiburg, Germany), and the results were expressed as the average percentage of control. IC50 values of DEBIO-025 and CsA were calculated by nonlinear curve fitting following the equation: Y = 100 − (YBottom × X/(IC50 + X)), where Y represents percentage inhibition and X represents the concentration of the agent. The viability of replicon cells was measured using the WST-8 cell counting kit according to the manufacturer's instructions (Dojindo, Kumamoto, Japan).
Western Blot Analysis of HCV NS3 and β-Actin.
HCV replicon cells (1 × 106) were lysed with 100 μl of lysis buffer (1% SDS, 0.5% Nonidet P-40, 150 mmol/l NaCl, 0.5 mmol/l EDTA, 1 mmol/l dithiothreitol, and 10 mmol/l Tris, pH 7.4). Five micrograms total protein was electrophoresed on a 10% SDS-polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA). Nonstructural protein 3 (NS3) of HCV was detected using the rabbit anti-NS3 (R212) polyclonal antibody that was prepared in our laboratory. Beta actin was detected using anti–β-actin monoclonal antibody (Sigma, St. Louis, MO).
Immunosuppressive Activity of DEBIO-025 and CsA by Interleukin-2 Reporter Gene AssayIn Vitro.
We examined the immunosuppressive activities of DEBIO-025 and CsA using a nuclear factor of activated T cells–dependent IL-2 reporter gene assay (Fig. 1B).19 We used Jurkat T-cells stably expressing lac-Z controlled by the IL-2 promoter. The cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 2 mmol/l glutamine, 50 μM 2-mercaptoethanol, and 100 U/ml hygromycin B. Jurkat T-cells were stimulated with phorbol-12-myristate-13-acetate (2.4 μM) and phytohemagglutinin (75 μg/ml) in the presence or absence of DEBIO-025 or CsA (10−9 to 2 × 10−5 mol/l). After incubation at 37°C for 20 hours, cells were harvested by lysis buffer (50 mmol/l Na2HPO4, pH 9.0, 10 mmol/l KCl, 1 mmol/l MgSO4, and 1% Triton X-100), and then β-galactosidase activity in the lysate was measured using 4-methyl umbelliferyl-β-D-galactoside (0.5 mmol/l; Sigma).
HCV Infection into Chimeric Mice.
We purchased chimeric mice from PhenixBio (Hiroshima, Japan). The chimeric mice were generated by transplanting human primary hepatocytes into severe combined immunodeficient (SCID) mice carrying the urokinase plasminogen activator transgene controlled by an albumin promoter.20 The chimeric mice used in this study were improved from the original ones, as described by Tateno et al.,21 and had a high substitution rate of human hepatocytes. Six weeks after hepatocyte transplantation, we intravenously injected each mouse with patient serum containing 106 copies of HCV genotype 1a (HCG9) or 1b (HCR6).22 HCV inoculations, drug administration, blood collection, and killing were performed under ether anesthesia. Blood samples were taken from the orbital vein and sera were immediately isolated. The protocols for animal experiments were approved by the local ethics committee. The animals received humane care according to NIH guidelines. Patients gave written informed consent before sampling.
Measurement of Human Serum Albumin.
Human serum albumin in the blood of chimeric mice was measured with a commercially available kit according to the manufacturer's instructions (Alb-II kit; Eiken Chemical, Tokyo, Japan).
Schedule for Administration of Agents into Chimeric Mice Infected with HCV Genotype 1b or 1a.
Treatment was started 12 weeks after HCV inoculation and continued during 14 days (Fig. 3A and Fig. 4A). Each treatment group comprised 3 animals. Peg-IFN and DEBIO-025 in mice with HCV genotype 1a or 1b were administered as follows: either Peg-IFN (30 μg/kg) was injected subcutaneously twice weekly alone or DEBIO-025 (100 mg/kg) was given orally every day alone, or a combination of both drugs was given. CsA (100 mg/kg) was given orally every day combined with Peg-IFN (30 μg/kg) subcutaneously twice weekly only to chimeric mice inoculated with genotype 1a.
Measurement of HCV Core Protein in Liver.
Liver tissues were homogenized in lysis buffer (10 mM Tris pH 7.5, 1% SDS, 0.5% NP-40, and 150 mM NaCl) and centrifuged for 60 seconds at 16,000 g. HCV core protein was quantified using a commercially available kit (Ortho Clinical Diagnostics, Tokyo, Japan).23
Quantification of HCV RNA by Real-Time Reverse Transcription PCR.
HCV RNA in serum or liver tissue was extracted using the acid guanidinium-phenol-chloroform method. Quantification of HCV RNA was performed using real-time reverse transcription PCR based on TaqMan chemistry, as described.24
Liver tissues obtained from mice were embedded in OCT compound (Ted Pella, Redding, CA). The frozen tissues were cut into thin sections (6 μm) and placed on glass slides. The sections were fixed in 10% buffered formalin and then treated with 0.1% Triton X-100. To detect HCV protein, the slides were incubated with rabbit anti-core protein IgG and then donkey anti-rabbit IgG polyclonal antibody [Fab fragment, labeled with horseradish peroxidase; Dako, Glostrup, Denmark]. The horseradish peroxidase label was amplified with FITC-conjugated tyramide according to the manufacturer's instructions (Molecular Probes, Eugene, OR). To detect human hepatocytes, liver sections were probed by anti-human hepatocyte monoclonal antibody (Dako), followed by anti-mouse IgG-Alexa 546 (Molecular Probes). Nuclei were stained by DAPI (Molecular Probes). Normal rabbit IgG was used as a control.
Antiviral Activity of DEBIO-025 in HCV Subgenomic Replicon Cells.
The anti-HCV effects of DEBIO-025 and CsA were initially confirmed using HCV replicon cells. Both inhibited the replication of HCV replicon RNA in a concentration-dependent manner. The IC50 values of DEBIO-025 and CsA against replicon cell line of FLR3-1 were 0.06 μg/ml and 0.31 μg/ml respectively (Fig. 2B). The IC50 values of DEBIO-025 and CsA against replicon cell line of R6FLR-N were 0.07 μg/ml and 0.27 μg/ml, respectively (Fig. 2C). The inhibitory effect of DEBIO-025 was approximately 5-fold greater than that of CsA. When cell viabilities were monitored using WST-8, DEBIO-025 differed from CsA by showing a reduction of cell viability only in R6FLR-N cells (CsA reduced cell viability in both types of replicon cells; Fig. 2D-E). In R6FLR-N cells, DEBIO-025 at 3.33 μg/ml reduced cell viability by an average of 27.8%, whereas CsA at the same concentration reduced cell viability by an average of 57.2% (Fig. 2E). Western blotting of FLR3-1 cells showed that expression levels of NS3 protein, but not β-actin, were decreased by treatment with DEBIO-025 or CsA (Fig. 2F).
Immunosuppressive Activity of DEBIO-025.
To examine the immunosuppressive activity of DEBIO-025, we used an nuclear factor of activated T cells–dependent IL-2 reporter gene assay. DEBIO-025 showed only a slight inhibitory effect on this system, with an activity that was 7,000-fold lower than that of CsA (data not shown). This indicates that the substitution of 2 amino acids in CsA to produce DEBIO-025 resulted in a greatly reduced immunosuppressive activity.
Human Albumin Levels in Mouse Serum After Transplantation of Human Hepatocytes.
The concentration of human albumin in the serum of the chimeric mice was measured to provide an index of the substitution rate of mouse to human hepatocytes after transplantation.21 The concentration measured 20 days after transplantation of human hepatocytes was 3.5 to 6.0 mg/ml, indicating that human hepatocytes had settled into the chimeric mice. At 6 weeks after transplantation, we inoculated the mice with patient serum containing HCV genotypes 1a or 1b. We repeatedly measured the concentrations of human albumin after inoculation and found that they reached a plateau at approximately 6.5 mg/ml. Although the mice were infected with HCV, significant reductions of the human albumin concentrations were not observed (Fig. 5A-B).
Persistent Infection of HCV in Chimeric Mice.
To determine whether the chimeric mice were persistently infected with HCV, we measured HCV RNA levels in serum weekly after the inoculation. HCV RNA disappeared at the first week and was then detected from 2 weeks after the inoculation. Four weeks after infection, HCV RNA levels reached 108 to 109 copies/ml in the genotype 1a group (Fig. 5C) and 106 to 107 copies/ml in the genotype 1b group (Fig. 5D). These results showed that our patient sera containing HCV had infected the chimeric mice. Furthermore, the increase of HCV levels in the serum was time dependent, indicating that HCV replicated and accumulated in the human hepatocytes of the chimeric mice.
Effect on HCV RNA Levels of DEBIO-025 and/or Peg-IFN in Mice Infected with HCV Genotype 1b.
DEBIO-025 alone did not inhibit HCV replication, but Peg-IFN alone reduced serum HCV RNA levels approximately 10-fold from day 3 to day 14 (Fig. 3B). A 100-fold reduction was observed with the combined treatment (Fig. 3B). These results indicated an effect of DEBIO-025 that appeared to be synergistic with Peg-IFN against genotype 1b. The concentration of human serum albumin and the body weight of the mice did not change significantly during this period (Fig. 3C, D). After cessation of treatment, HCV RNA levels returned to107 copies/ml.
Comparison of DEBIO-025 and CsA Effect in Chimeric Mice Infected with HCV Genotype 1a.
The serum HCV RNA levels with the administration of DEBIO-025 or Peg-IFN alone seemed to be similar at day 7 and at day 14 as compared with those seen in mice infected with genotype 1b (Fig. 4B). The combined administration of DEBIO-025 with Peg-IFN resulted in a 600-fold reduction of HCV RNA levels at day 14 (Fig. 4B). The combined administration of CsA and Peg-IFN resulted in the death of all treated mice within 4 days. The body weight of all CsA-treated mice was reduced by more than 20% during this period (Fig. 4C). The concentration of human serum albumin in the mice treated with CsA did not change significantly (data not shown). This toxicity was not observed with DEBIO-025 and Peg-IFN.
Quantification of Hepatic HCV RNA and Core Protein Levels and Immunohistochemistry at the End of Treatment in Chimeric Mice Infected with Genotype 1a.
At the end of treatment, hepatic HCV RNA was quantified by real-time reverse transcription PCR, and core protein levels were quantified by enzyme-linked immunosorbent assay (Fig. 6A,B). DEBIO-025 monotherapy (1a-3 mouse) reduced HCV RNA by 3-fold compared with the nontreated mouse (1a-4 mouse). Peg-IFN reduced both HCV RNA and core protein levels by approximately 10-fold (1a-2 mouse). Combined treatment with DEBIO-025 and Peg-IFN resulted in an approximately 100-fold reduction in HCV RNA and HCV core protein levels (1a-1 mouse). Moreover, immunohistochemistry was performed. In 1a-4 mouse, HCV core protein was detected in human hepatocytes. In 1a-1 mouse, HCV core protein was not detected by immunohistochemistry; however, reduced HCV core protein was quantified by enzyme-linked immunosorbent assay, which is more sensitive than immunohistochemistry (Fig. 6C, D).
Development of new anti-HCV drugs has been significantly impeded by the lack of a suitable cell culture model for the propagation of HCV in laboratories. This obstacle has been partially overcome by the development of the replicon system, which can be used for evaluating the in vitro anti-HCV effect of compounds. However, because adaptive mutation into the replicon genome and host permissiveness enable particularly efficient replication in cultured hepatoma cell lines,25 evaluation of HCV drugs using replicon systems alone is considered insufficient. The only animal species readily infected with HCV has been the chimpanzee, which is labor-intensive and expensive to use, and is associated with ethical problems. The chimeric mouse with human hepatocytes has recently been developed as a practical small animal model that can be infected with HCV.20 This model is promising for the evaluation of new anti-HCV drugs because the mice are easy to handle, grow rapidly, and are well characterized genetically and immunologically. In this study, we used chimeric mice to bridge the gap between the replicon system and naive HCV replication in human liver, and to examine the anti-HCV effect of DEBIO-025, a novel cyclophilin inhibitor and non-immunosuppressive cyclosporin.
We found that HCV from our patient sera were able to infect the chimeric mice and persistently replicate over several weeks. HCG9 (1a) and HCR6 (1b) reached 108 to 109 copies/ml and 106 to 107 copies/ml, respectively, resulting in HCV RNA levels in serum that were higher than those previously reported.20 This was probably because of a high substitution rate of human hepatocytes in the chimeric mice. When Mercer et al.20 initially developed chimeric mice infected with HCV, they reported that human albumin concentrations in sera of the mice reached 2 mg/ml and that the substitution rate of liver from mouse to human was approximately 50%. In our study, the human albumin concentration in the chimeric mice reached 6.5 mg/ml, which would be consistent with a higher substitution rate of 80% to 90%.21 In addition, our findings also indicate that the plateau point of HCV RNA in serum depends on the type of inoculum, because the HCV RNA levels were different for HCG9 and HCR6. Taken together, the results suggest that our chimeric mice propagated large amounts of HCV in their livers.
Although DEBIO-025 strongly inhibited replication of the HCV replicon, it did not affect the replication of naive HCV in vivo when given as monotherapy. These results probably indicate differences between the replication of naive HCV in vivo and the replicon system. The sensitivity of HCV strains to CsA and non-immunosuppressive cyclosporins was variable, depending on their cyclophilin requirement for their replication.26 Cyclophilin polymorphism and its role in HCV replication will be the focus of future study.
The HCV RNA levels are known to decline biphasically in most patients treated with IFN.27 During the first phase, there is a rapid drop in viremia that reflects the direct inhibition of HCV replication. During the second phase, there is a slower decline in serum HCV RNA levels, which appears to reflect the elimination of infected cells by host immune responses. In chimeric mice, the second-phase decline is not obvious, because they lack T cells and B cells (being SCID). Thus, it appears that DEBIO-025 accelerates the decline in HCV RNA levels induced by Peg-IFN during the first phase. There is no evidence that DEBIO-025 enhances the interferon pathway. Also, recent in vitro findings show that cyclosporins do not modify the IFN-α signal transduction pathway as assessed by 2′, 5′-oligoadenylate synthetase (2′, 5′-OAS) levels.28 It therefore seems likely that the apparent synergistic effect of DEBIO-025 seen in our in vivo model is not solely related to the antiviral effect mediated by IFN. The DEBIO-025 inhibition of cyclophilin may produce a proper anti-HCV effect by interacting with the RNA-dependent RNA polymerase.11
CsA was originally used as an immunosuppressive agent, and we previously demonstrated in clinical trials that CsA has an anti-HCV effect.9 However, CsA is not devoid of adverse effects, such as hypertension, neurotoxicity, and nephrotoxicity, limiting its therapeutic usefulness against HCV.29 The immunosuppressive action of CsA occurs by inhibition of calcineurin. Our findings showing that DEBIO-025 exhibits a 7,000-fold lower immunosuppressive activity than CsA suggest that it has less affinity to calcineurin and may lead to fewer adverse effects in patients.
In conclusion, our results indicate that naïve HCV replication in vivo is inhibited by the combined administration of the cyclophilin inhibitor DEBIO-025 and Peg-IFN. These findings support further evaluation of DEBIO-025 as a promising drug for the treatment of chronic hepatitis C.
The authors thank Isao Maruyama and Hiroshi Yokomichi of PhenixBio Co., Ltd. for the maintenance of the chimeric mice.