Virus–Drug Interactions—Molecular Insight Into Immunosuppression

PERSPECTIVES

OPINION studies on different aspects of the viral life cycle and pathophysiology, including viral Virus–drug interactions—molecular entry, trafficking in the cell and assem- bly and release of de novo virus particles.8 insight into immunosuppression and HCV These models have enabled basic research to make considerable contributions to an Qiuwei Pan, Hugo W. Tilanus, Herold J. Metselaar, Harry L. A. Janssen improved understanding of how immuno- and Luc J. W. van der Laan suppressive compounds can actually influence HCV infection and the antiviral action Abstract | Liver transplantation is an effective treatment for end-stage liver disease of interferons. that is attributable to chronic HCV infection. However, long-term outcomes are In this article, we explore the potential compromised by universal virological recurrence in the graft. Reinfection that molecular interaction of different immuno occurs after transplantation has increased resistance to current interferon- suppressants with HCV and interferon based antiviral therapy and often leads to accelerated development of cirrhosis. signalling. We also discuss how to extrapo- Important risk factors for severe HCV recurrence are linked to immunosuppression. late these laboratory findings into clinical Owing to the lack of good randomized, controlled trials, the optimal choice of practice and drug development. immunosuppressants is still debated. By contrast, much progress has been made in Calcineurin inhibitors the understanding of HCV biology and the antiviral action of interferons. These new insights have greatly expanded our knowledge of the molecular interplay between The primary cellular targets of the calci neurin inhibitors are immunophilins. HCV and immunosuppressive drugs. In this article, we explore the effect of different Ciclosporin A binds to cyclophilins, and immunosuppressants on the complex cellular events involved in HCV infection tacrolimus (also known as FK506) binds and interferon signalling. Potential implications for clinical practice and future drug to FK binding proteins (FKBPs); both development are discussed. events result in a profound inhibition of Pan, Q. et al. Nat. Rev. Gastroenterol. Hepatol. 9, 355–362 (2012); published online 17 April 2012; the phosphatase activity of calcineurin. doi:10.1038/nrgastro.2012.67 Immunophilin-dependent signal transduction via calcineurin represents a key event Introduction medication.5 Over the past five decades, the in the activation of T‑cell proliferation End-stage liver diseases that are caused by immunosuppressive regimen has evolved by regulating expression of the gene that chronic HCV infection are currently the from being mainly based on glucocortico encodes IL‑2 (Figure 1a). Apart from their leading indications for liver transplanta- steroids, azathioprine and ciclosporin A to role in calcineurin signalling, immuno tion.1 However, HCV reinfection of the steroid-sparing regimens involving anti- philins are catalysts of protein folding and graft occurs universally and often results IL‑2 receptor antibody induction therapy, as such have a supportive role in viral infec- in accelerated recurrence of liver fibrosis calcineurin inhibition by tacrolimus tion. Viruses, including HIV, herpes simplex and early development of cirrhosis.2 HCV and the use of new-generation immuno virus, vaccinia virus, vesicular stomatitis infection is also the most frequent cause suppressants—mycophenolate mofetil and virus and coronaviruses, take advantage of of liver disease after kidney transplanta- rapamycin. The changes in drug types and immunophilins for their replication, and tion and is an independent risk factor for drug combinations together with increases this virus–host interaction is inhibited by graft loss.3 In general, the current standard in the age of donors have probably contrib- ciclosporin A.9,10 Moreover, cyclophilin A therapy for patients with HCV (PEG-IFN‑α uted to the reported worsening of HCV can actually incorporate itself into these in combination with ribavirin) is much less recurrence since the early 1990s.6 Although viral particles.9,11 For HCV, it is well estab- effective in patients who have had a liver enormous efforts have been devoted to lished that cyclophilins have an essential transplant than in those who have not clinical research,7 the choice of immuno- role in viral replication and de novo virus had a transplant.4 An aggravated course of suppressants in HCV-positive recipients is production. Early studies suggest that HCV infection and increased resistance to anti still a subject of debate. replication is dependent on the interaction viral therapy have been attributed to several The lack of good model systems ham- between cyclophilin B and nonstructural host and viral factors, in particular the pered HCV research in its early stages. protein 5B (NS5B, HCV RNA polymerase) application of specific immunosuppressive However, this deficiency has improved to stimulate its RNA binding activity and dramatically in the past decade with the thereby promote the de novo synthesis of development of various in vitro systems positive and negative stranded RNA.12,13 Competing interests of cell culture (Box 1). The HCV pseudo Ciclosporin A blocks the binding of cyclo- H. J. Metselaar declares associations with the following companies: Astrellas, Novartis. See the particles, the HCV replicons and the JFH‑1- philin B to NS5B RNA and thereby inhib- 12–14 article online for full details of the relationships. derived infectious HCV cell culture systems its viral replication. Cyclophilin A The other authors declare no competing interests. have all demonstrated their importance in also directly interacts with NS5B15 and is

Box 1 | HCV culture models intriguing issue. Hirano et al.28 suggested trials and the first results are encourag- ■■ The first full-length HCV clones could that tacrolimus, but not ciclosporin A, ing. In a cohort infected with HCV and infect chimpanzees but replication interferes with interferon signal transduc- HIV, 2 weeks of treatment with Debio‑025 in vitro remained ineffective. By creating tion. However, a later study did not confirm decreased HCV viral load by more than antibiotic-resistant HCV genomes, this suggestion, instead demonstrating in 3‑log, including in patients infected with selection of replication-competent viral two state-of-the-art cell culture models that HCV genotypes 1, 3 and 4. By contrast, clones became possible. This advance tacrolimus does not interfere with either the anti-HIV effects were much less pro- led to the development of subgenomic or interferon signalling or the antiviral activity nounced (1‑log reduction of viral load).34 bicistronic (two cistrons) replicons, which 29 mimic HCV replication. HCV replicons of interferon. Therefore, no further mecha- In treatment-naive patients infected with are most efficient in a human hepatoma nistic understanding has been unveiled on just HCV, treatment with Debio‑025 alone cell line (Huh7), and can be linked to this issue. resulted in a more than 2‑log reduction of fluorescent or luminescent reporter genes In fact, the first in vitro evidence that viral load in those with genotypes 1 and 4, ■■ Early studies of HCV viral entry used ciclosporin A but not tacrolimus can inhibit and a more than 4‑log reduction in those pseudoparticles incorporating HCV HCV replication14 sparked the clinical with genotypes 2 and 3.35 When combined envelope proteins E1 and E2 onto a debate on the possible differential effect of with PEG-IFN‑α and ribavirin, additive retrovirus or lentivirus these two drugs on HCV recurrence.30 As antiviral effects were demonstrated.35 ■■ The highly effective HCV infectious model was based on the first genotype 2a clone HCV only infects humans and chimpanzees, called JFH‑1, isolated from a Japanese small animal models for HCV infection are Glucocorticosteroids patient with fulminant hepatitis C. JFH‑1- still difficult to create. One of the few estab- Glucocorticosteroids have been used since derived HCV models can be used to study lished experimental animal models to study the early years of organ transplantation. the complete viral life cycle and chimeric HCV infection is the SCID/uPA (severe Prednisolone and its close analogue dexa- genomes have been constructed of all combined immunodeficient, urokinase methasone are potent suppressors of the seven known genotypes plasminogen activator transgenic) mouse immune system, as they modulate cellular engrafted with primary human hepatocytes. and inflammatory responses via stimula- involved in modulating the polyprotein A study published in 2011 by the group that tion or inhibition of gene transcription cleavage activity of NS2.16 In addition, pioneered the development of this mouse (Figure 1).36 Although evidence indicates cyclophilin A interacts with NS5A and model has attempted to define the effect that the oestrogen receptor is functionally stimulates its binding with HCV RNA.15,17 of ciclosporin A and tacrolimus on serum involved in HCV replication by promoting As ciclosporin A interacts with both HCV titres and antiviral interferon therapy NS5B association with the viral replication cyclophilin A and cyclophilin B, it is con- in vivo.31 Although no statistically signifi- complex,37 the role of other glucocortico ceivable that it affects multiple steps in the cant differences were observed between steroid receptors is largely unknown. A virus life cycle (Figure 1b). The anti-HCV ciclosporin A and tacrolimus either on study using the HCV subgenomic replicon activity of ciclosporin A is not mediated by HCV viral titres or the efficacy of interferon model38 found that both prednisolone and an interferon response and is independ- therapy, effects could have been masked by dexamethasone have no stimulatory effects ent of calcineurin signalling.14 Although some technical limitations. Firstly, the base- on RNA levels, but rather have minor inhib- studies showed a role for FKBP8 in HCV line HCV titres differed between the tested itory effects.39 Using an infectious HCV replication18 through interaction with groups, with a trend toward higher base- model (the JFH1-derived chimera Jc1 in NS5A,19 FKBP8 lacks several amino acid line HCV RNA levels in the ciclosporin A Huh‑7.5 cells), similar results were obtained residues thought to be important for tac- arms than in the tacrolimus arms. Secondly, showing ~50% inhibition of viral replication rolimus binding.18,20 No evidence exists that the antiviral response to interferon in this at high doses of prednisolone.40 However, tacrolimus inhibits HCV replication.14 model was much less robust than that in this infectious model, prednisolone Several in vitro studies have shown observed in patients. Moreover, models stimulated HCV infection by enhancing that ciclosporin A can act in synergy with in immunodeficient mice cannot evalu- virus entry.40 This specific enhancement IFN‑α,21–24 but clinical evidence to support ate indirect effects of immunosuppressive was mechanistically linked to the upregu- these findings is still limited.25 The results of drugs on antiviral immune responses unless lation of the two essential HCV entry a prospective, randomized, controlled pilot one of the latest immunocompetent mouse factors—occludin and scavenger receptor study published in 2010 demonstrated that models is used.32 class B type I—at both mRNA and protein a switch from tacrolimus to ciclosporin A The development of ciclosporin A deriva- levels.40 On the basis of their observations, resulted in a modest drop in serum levels tives strongly indicates that ciclosporin A the authors speculated that treatment of HCV RNA and an enhancement of the would have anti-HCV activity both in vitro with a steroid bolus (>250 mg predniso- response to antiviral interferon therapy in and in patients. Three cyclophilin inhibitors lone per day) in patients with HCV might patients who had received a liver transplant.26 are currently being evaluated in preclinical foster virus dissemination through facilita- This finding supports those of a previous and clinical studies: Debio‑025, NIM811 tion of virus entry into hepatocytes, thus study that observed a benefit of converting and SCY‑635.33 These compounds are aggravating HCV recurrence. Moreover, from tacrolimus to ciclosporin A during developed by structural modifications of treatment with prednisolone probably sup- pre-emptive IFN‑α and ribavirin antiviral ciclosporin A to enhance the antiviral activ- presses the patient’s immune control of the therapy in patients who were HCV positive ity and reduce the immunosuppression cap HCV-infected cells, by effects on T cells41 and had undergone liver transplantation.27 acity by increasing cyclophilin binding and and plasmacytoid dendritic cells,42 and The mechanism by which ciclosporin A can abolishing calcineurin affinity, respectively. thereby might indirectly accentuate infec- promote the response to interferon is an Debio‑025 has been the first to enter clinical tion.39 Plasmacytoid dendritic cells are

Figure 1 | Mechanisms of action of GCs and a ◀ CaN inhibitors. Mechanisms are shown in a | leukocytes and b | hepatocytes. GCs diffuse into cells and bind to the GR. The GC– NFκB AP1 NF-ATc GR complex translocates to the nucleus to GR GRIP1 GRIP1 GR IL-2 regulate gene expression. The GC–GR Nucleus complex interacts with GRE promoter GC Cytokines GC elements and suppresses the expression of Chemokines inflammatory genes and cytokines, such as Cytoplasm IL‑2, thereby inhibiting T‑cell proliferation. P Steroids facilitate HCV entry in hepatocytes by upregulating gene expression of two NF-ATc essential HCV entry receptors—occludin and GR P Tac scavenger receptor class B type I— CaN GC FKBP conceivably via positive GRE promoter 2+ Cell proliferation Ca CsA elements. GR and type I interferon signalling GC CyP share the coregulator GRIP1. Ciclosporin A binds to cyclophilin, while tacrolimus binds to FK506-binding protein, forming a complex to TCR/CD3 block calcineurin. Suppression of the serine/ threonine phosphatase activity of calcineurin results in inhibition of TCR/CD3 induced T‑cell proliferation by blockage of IL‑2 production. GC Ca2+ Ciclosporin A binds to cyclophilin A and MHC/peptide CsA Tac cyclophilin B to block the function of NS2, NS5A or NS5B to affect viral replication in hepatocytes. Protein phosphorylation induced b IFN-α/β LD by signal transduction is indicated by P in orange. Abbreviations: AP1, activator protein 1; CaN, calcineurin; CsA, ciclosporin A; CyP, cyclophilin; FKPB, FK506- HCV entry binding protein; GC, glucocorticosteroid; GR, cytoplasmic receptor; GRE, glucocorticoid response element; GRIP1, glucocorticoid receptor-interacting protein 1, IRF9, interferon SB-R1 CD81 regulatory factor 9; ISG, interferon-stimulated gene; ISRE, interferon stimulated response CsA Tac element; JAK1, Janus kinase 1; LD, lipid TYK2 GC P FKBP droplet; NF‑ATc, nuclear factor of activated JAK1 ST CyP S TAT

AT T cells; NF B, nuclear factor B; NS, P HCV κ κ 2

1 nonstructural protein; SR‑BI, scavenger P P GR receptor class B type I; STAT, signal transducers and activators of transcription; GC NS5B NS2 Tac, tacrolimus; TCR, T‑cell receptor; TYK2, NS5A nonreceptor tyrosine-protein kinase. Cytoplasm Viral replication

GC immunosuppressants are used as mainte- GR nance immune suppression after transplan- Nucleus tation. In cases of acute cellular rejection GRIP1 episodes, patients receive several boluses of P GR target GRIP1 ST ISGs genes methylprednisolone to reverse the rejection. AT2 GC GC ST The most compelling evidence that steroids P AT1 GR GR affect HCV viral load actually came from IRF9 studies in patients with HCV who did not undergo a liver transplant.44–46 In a trans- ISRE GRE plant setting, the general consensus is that steroid avoidance47 or slow tapering of the dose48 is associated with reduced disease of particular interest in HCV infection, abolishes the anti-HCV activity of plasma- recurrence, whereas boluses for treat- as they produce large amounts of IFN‑α; cytoid dendritic cells in vitro (P. E. de Ruiter, ing acute rejection can increase the viral however, the capability of plasmacytoid personal communication). load.49,50 However, a randomized multi dendritic cells to produce IFN‑α seems to In liver transplantation, glucocortico centre study comparing steroid-free therapy be reduced in patients with chronic HCV.43 steroids are often given as an induc- and standard immunosuppression showed Experimental data from our group have tion protocol during surgery and no clear advantage of not using steroids in shown that treatment with prednisolone low doses in combination with other liver transplant recipients who had HCV.51

Two systematic Cochrane reviews with decreases intracellular levels of guanosine point of time. However, the in vivo effi- meta-analyses of randomized, controlled nucleotide pools resulting in inadequate cacy of many antiviral compounds (such trials have been performed in the settings quantities for nominal DNA duplication, as ribavirin) can be observed only when of chronic HCV52 and HBV53 infection which was thought to be the antiprolifera- combined with other antivirals. Ribavirin to comprehensively assess the effects of tive and immunosuppressive mechanism has shown substantial anti-HCV activity glucocorticosteroids on interferon antiviral of MPA (Figure 2a).64 This mechanism is in vitro but is generally considered to have therapy. No statistically significant effect of probably also responsible for its antiviral little or no detectable antiviral activity as a steroids on the virological response at the effects against at least the West Nile,23 yellow monotherapy in patients.70 However, when end of treatment was found in patients fever61 and Chikungunya viruses,62 as sup- combined with IFN‑α, ribavirin increases with HCV.52 By contrast, pretreatment with plementation by exogenous guanosine the sustained virologic response by three- steroids was associated with a considerably almost completely overcomes these inhibi- fold.71 Similarly, a previous clinical study higher frequency of loss of hepatitis B e tory effects. By contrast, supplementation showed that combined treatment with antigen and HBV DNA, although no statis- of guanosine had little effect on the inhibi- MMF and PEG‑IFN‑α in patients with tically significant effect on clinical outcomes tion of HCV replication.23,65 Consistently, chronic HCV resulted in a considerably was found.53 However, mechanistic studies ectopic expression of IMPDH2 mutants improved end-of-therapy response rate in have shown that glucocorticosteroids can lacking the binding site for MPA largely a difficult-to-treat population of previous interfere with signal transduction of the restored its antiproliferative effect, but with nonresponders to standard therapy.72 The interferon receptor. The glucocorticosteroid only minor effects on HCV replication, sug- sustained virologic response of the IFN‑α receptor and type I interferon receptor sig- gesting other mechanisms are involved in its and MMF combination group, however, was nalling pathways share the cofactor gluco anti-HCV action.66 Unexpectedly, MPA was lower than that of the group treated with corticoid receptor-interacting protein 1 found to induce the expression of impor- IFN‑α and ribavirin.72 A possible explana- (GRIP1).54,55 Glucocorticosteroids sup- tant antiviral interferon-stimulated genes tion for these limited sustained virologic press interferon responses by antagonizing in HCV cell culture models, including responses is that MMF interferes with a the heterotrimeric STAT1–STAT2–IRF9 interferon regulatory factor (IRF) 1, IRF9 robust antiviral immune response required (ISGF3) activity via GRIP156 or attenuation and interferon-induced transmembrane to eliminate HCV.23 Notably, MPA works of STAT1 activation through induction of protein 3 (IFITM3).66 Using an RNAi- in synergy with IFN‑α on HCV replication SOCS1, which is a suppressor of cytokine based loss-of-function approach, IRF1 was in vitro.23 When combined with IFN‑α, signalling 1 (Figure 1).57 However, these demonstrated to be directly involved in the MPA augments the transcription of mul- events probably differ between cell types anti-HCV activity of MPA.66 tiple interferon-stimulated genes that are and no direct link between these molecular The expression of a panel of interferon- mediated by interferon-stimulated response interactions and viral infection has been stimulated genes was also considerably elements.66 These latest findings have shed well established. Therefore, it is of particular higher in peripheral blood mononuclear some light on the molecular basis of how relevancy to further investigate the actual cells of patients who had received a kidney MPA works in synergy with IFN‑α. effects of steroids on interferon antiviral transplant and were maintained on MMF response in HCV models. immunosuppression, compared with Rapamycin peripheral blood mononuclear cells of Rapamycin, a new-generation immuno- MMF and MPA healthy control individuals.66 Similar results suppressant, has been gaining increas- Mycophenolic acid (MPA) is the activated have been reported for ribavirin, which ing favour in the transplantation context, form of the prodrug mycophenolate mofetil also induces expression of a broad spec- mainly attributable to its low nephrotoxicity (MMF). MMF and MPA are part of the trum of interferon-stimulated genes.67,68 and potential anticancer properties.73 class of antimetabolite immunosuppressive Interestingly, like MPA, ribavirin is a known Rapamycin engages the cytosolic protein agents that also includes azathioprine. The inhibitor of IMPDH.69 Inhibiting IMPDH FKBP12 to form a complex. This complex effect of azathioprine on HCV has not been and inducing the expression of interferon- inhibits the mammalian target of rapamycin extensively studied, despite one early study stimulated genes probably simultaneously (mTOR) pathway by directly binding to the suggesting that this agent has antiviral contribute to the anti-HCV action of MPA mTOR complex 1, resulting in blockage of effects on two Flaviviridae viruses.58 (Figure 2b). Whether IMPDH by itself is cell cycle progression at the G1 to S phase MMF is commonly used in kidney involved in the induction of the expres- and thereby causing inhibition of T‑cell transplantation, but it is also approved by sion of interferon-stimulated genes is not proliferation (Figure 2). the FDA for the prophylaxis of allograft fully clear. However, supplementation with One particular interesting feature of rejection after heart or liver transplanta- exogenous guanosine did not interfere with rapamycin is that it induces autophagy by tion.59 In addition to its potent immuno inducing the expression of these genes.66 inhibition of mTOR. Autophagy is a process suppressive capacity, MPA also has a broad Although the safety and efficacy of MMF for catabolising organelles and other cyto- spectrum of antiviral activity in vitro as an immunosuppressive medication in plasmic components to balance cellular against numerous DNA and RNA viruses, patients with HCV who received a trans- metabolism and to promote cell survival including dengue virus,60 West Nile virus,23 plant has been demonstrated, the exact during stressful conditions. Autophagy is yellow fever virus,61 Chikungunya virus,62 effects on HCV recurrence have not been also an important event in regulation of the HBV63 and HCV.23 MPA is an uncompeti- clearly studied in prospective, randomized cellular response against viral infections.74 tive inhibitor of inosine monophosphate and double-blinded trials. Results from The role of autophagy is complex and can dehydrogenase (IMPDH), in particular the prospective nonrandomized or retrospec- have both antiviral and proviral effects in isoform IMPDH2. Inhibition of IMPDH tive studies remain controversial at this different virus infections. During infection,

◀ Figure 2 | Mechanisms of action of a mycophenolic acid and rapamycin. Mechanisms shown in a | leukocytes and b | hepatocytes. The uptake of MPA by cells involves organic anion transporting Autophagy Nucleus polypeptides and probably other transporters as well. MPA is an uncompetitive inhibitor of IMPDH and results in the inhibition of de novo IMP mTORC1 mTORC2 nucleotide biosynthesis. Insufficient GMP intracellular levels of guanosine nucleotide pools result in a defect of nominal DNA IMPDH duplication that is thought to be the Cell proliferation immunosuppressive mechanism of MPA. In FKBP hepatocytes, persistent attenuation of nucleotide biosynthesis by MPA partially contributes to the inhibition of HCV Cytoplasm replication. In addition to this IMPDH- dependent antiviral pathway, MPA also conveys a rapid antiviral effect mediated by induction of the expression of interferon- MPA Rapamycin stimulated genes. Through an unknown mechanism, MPA directly potentiates the activity of the ISRE promoter element both in b IFN-α/β the presence or absence of IFN‑α stimulation. Rapamycin engages the cytosolic protein MPA Rapamycin FKBP12 to form a complex. This complex inhibits the mTOR pathway by directly binding to the mTORC1, resulting in blockage of cell cycle progression at the G1 to S phase and thereby causing inhibition of T‑cell proliferation. Rapamycin is a potent Cytoplasm autophagy inducer, which facilitates HCV infection. The mTOR pathway directly interacts FKBP TYK2 with interferon-activated JAK–STAT signalling. P Protein phosphorylation induced by signal JAK1 S GMP ST IMPDH TAT ? transduction is indicated by P in orange. P AT 2

1 Abbreviations: FKBP12, FK506 binding P P mTORC1 protein 12; GMP, guanosine monophosphate; IMP HCV IMP, inosine monophosphate; IMPDH, inosine monophosphate dehydrogenase; IRF9, Autophagy mTORC2 ? interferon regulatory factor 9; ISG, interferon- stimulated gene; ISRE, interferon stimulated Nucleus response element; JAK1, Janus kinase 1; MPA, mycophenolic acid; mTOR, mammalian target of rapamycin; mTORC, mammalian P ST ISGs target of rapamycin complex; STAT, signal AT2 ST transducers and activators of transcription; P AT1 TYK2, nonreceptor tyrosine-protein kinase. IRF9

ISRE infection by reducing the innate immunity of host cells, including interferon responses79,80 Indeed, accumulating evidence autophagy can be a surveillance mechanism Several studies have shown that HCV exists for a direct link between the mTOR that delivers viral antigens to endosomal infection induces autophagy in hepato- signalling pathway and interferon-activated compartments to stimulate innate immune cytes via the unfolded protein response.76,77 JAK-STAT signalling.81–83 Type I interferon signalling and to provide processed anti- However, the induced autophagy induces phosphorylation and activation of gens for presentation in the major histo- appears incomplete, as the maturation mTOR in a PI3-kinase-dependent manner.83 compatibility complex. Virus infection can of autophagosomes to autolysosomes is In addition, mTOR complex 1 was found to trigger the induction of autophagy, but can blocked. Therefore, autophagosomes are stimulate the production of type I inter also block the function of the autophagy not degraded but instead support viral ferons through activation of IRF5 and pathway or even subvert autophagy. By replication.77 HCV viral proteins, such IRF7.82 On the basis of these mechanistic contrast, viruses can directly exploit the as NS5B, directly interact with the host insights, it is conceivable that rapamycin autophagy machinery to facilitate replica- proteins that are required for the induc- could affect HCV recurrence and antiviral tion or to promote their maturation and the tion of autophagy.78 In addition, induction interferon therapy, though clinical evidence dissemination of virions.75 of autophagy by HCV seems to facilitate to support this theory is still limited.84,85

Novel immunosuppressants TYK2.93 These pathways have considerable lead to serious or even life-threatening Despite the success of current immuno implications in both defence of virus infec- adverse effects. Therefore, the interac- suppressive agents in reducing the incidence tion and interferon responses. In addition, tion of new antiviral compounds and of acute cellular rejection, the toxicity asso- expression and activation of JAK3, which immunosuppressants requires extensive ciated with these regimens has now become is involved in defence against dengue virus studies of drug–drug interactions before a major obstacle for positive long-term out- infection, have been reported in hepatic designing any antiviral therapy trial in the comes of liver transplantation. Currently, cells.94 Therefore, studying the effects post-transplantation setting. novel agents, including small molecules, are and mechanisms of CP‑690550 on both In summary, progress in understanding at various stages of clinical development.86 HCV infection and interferon signalling HCV biology is important in evaluating These novel small molecules include the would be highly relevant. the complex effects of immunosuppressive new generation ciclosporin A analogue, medication on HCV recurrence. Despite ISA247, the protein kinase C (PKC) inhibi- Conclusions clear effects of ciclosporin A and MPA in tor, AEB071, and the selective Janus kinase The development of subgenomic HCV HCV culture models, reliable clinical evi- inhibitor, CP‑690550. cell culture models has greatly contributed dence for their effect on HCV recurrence Given the evidence for an important role to the discovery of the antiviral effects of is still lacking. Inhibition of the immune of cyclophilins in the life cycle of HCV,12,15,16 ciclosporin A and MPA on viral repli- response against HCV by immuno investigation of the effects of ISA247 on cation. The use of a recently developed suppressants probably counteracts the HCV infection would be of particular inter- infectious culture model has revealed the direct antiviral effects. The effectiveness est. ISA247 only differs from ciclosporin A negative effects of steroids, specifically on of interferon-based antiviral therapy is by a single amino acid residue. The struc- HCV entry.40 However, a common ques- reduced after transplantation, but, to date, tural change induced by this substitution is tion from transplant clinicians is why no concrete evidence from HCV culture considered to result in a greater calcineurin these clear in vitro effects of HCV are not models that immunosuppressive drugs inhibitory potency and a more predict- univocally observed in clinical practice. interfere with interferon signal transduction able pharmacokinetic profile than ciclo- For instance, the clear antiviral activity of exists. Immunosuppressive medication for sporin A. An X‑ray crystal structure study ciclosporin A and MPA in vitro is yet to transplant recipients who have HCV should has revealed the basis for the cyclophilin A be confirmed in proper prospective clini- be further improved. The application of binding affinity and immunosuppressive cal studies, and retrospective data sketch a newly registered anti-HCV compounds potency of ISA247.87 As cyclophilin A is murky picture. One probable answer to this in transplant recipients should be care- a host factor that modulates infection of in vivo and in vitro discrepancy is that tam- fully tested because of potential concerns a number of viruses, it will be important pering of the host immune system against regarding adverse drug–drug interactions to study the potential antiviral activity of the virus masks the direct antiviral effects with immunosuppressants. ISA247 compared with ciclosporin A. of these immunosuppressive agents. The Although basic science alone will never AEB071 is a small-molecule inhibitor antiviral potency of particular immuno- resolve the clinical debate, it already pro- that targets multiple members of the PKC suppressants might not be strong enough vides mechanistic insight into how par- family. PKC inhibition results in decreased to result in a dramatic effect on viral load, ticular immunosuppressants can affect the T‑cell activation. Several members of the but could potentially be reflected in other course of infection and outcomes of anti PKC family have been linked to both HCV parameters, such as slowing down fibrosis viral therapy. The knowledge gained in a infection and interferon signalling.88,89 progression.95–98 Perhaps the potent effects liver-transplant setting will also definitely be Using cell culture models, however, no stim- of the derivates of ciclosporin A, such as a valuable reference for the management of ulatory effect of AEB071 on HCV infection Debio‑025, in patients were achieved by patients who are HCV positive and receive a was observed; instead, at high drug concen- both deprivation of immunosuppressive kidney transplant. Conceivably, these mech- trations a reduction in viral replication was properties and enhancement of cyclophilin anistic understandings will promote future found.90 This reduction might be related to binding affinity.34,35 investment in the initiation of randomized, inhibition of cell proliferation by AEB071. A major new milestone for HCV treat- controlled studies and development of Therefore, it was concluded in this study ment is the approval of two protease new-generation immunosuppressants for that AEB071 had no direct effect on HCV inhibitors by the FDA in 2011. Although HCV-positive transplantation. infection.90 Further investigation is required boceprevir and telaprevir are only approved to draw firm conclusions. in combination with PEG‑IFN‑α and riba- Department of Gastroenterology and Hepatology (Q. Pan, H. J. Metselaar, CP‑690550 was developed as a selective virin for treating adults with chronic HCV H. L. A. Janssen), Department of Surgery and inhibitor of JAK3, which is predominantly genotype 1 infection who have compen- Laboratory of Experimental Transplantation expressed in immune cells and is only sated liver disease, trials are expected to and Intestinal Surgery (H. W. Tilanus, bound by γ-chain-bearing cytokine recep- be designed to treat patients both before L. J. W. van der Laan), Erasmus MC‑University tors involved in the JAK–STAT signalling and after transplantation. As a caution, a Medical Center, sGravendijkwal 230, Room L458, 3015 CE Rotterdam, The Netherlands. pathway.91 Some of the immunosuppressive phase I study found that telaprevir inter- mechanism of CP‑690550 can be explained feres with the metabolism of both ciclo- Correspondence to: L. J. W. van der Laan as the result of IL‑2 inhibition, as JAK3 sporin A and tacrolimus by inhibition of [email protected] functions in the receptor signalling pathway cytochrome P450 3A enzymatic activity.99 for this cytokine.92 However, CP‑690550 has Telaprevir caused a tremendous increase in 1. Brown, R. S. Hepatitis C and liver transplantation. Nature 436, 973–978 (2005). also been shown to inhibit other members blood concentrations of both these calci 2. Yilmaz, N. et al. A prospective evaluation of of the JAK family, including JAK1, JAK2 and neurin inhibitors and could potentially fibrosis progression in patients with recurrent

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