Copper toxicity in Wilson disease explained in a new ...

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that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured .... RANTES receptors CCR1 and CCR5.6,7 Interestingly,. Met-CCL5 has no effect ...
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Anti-Chemokine Therapy for the Treatment of Hepatic Fibrosis: An Attractive Approach Berres ML, Koenen RR, Rueland A, Zaldivar MM, Heinrichs D, Sahin H, et al. Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice. J Clin Invest 2010;120:4129-4140. (Reprinted with permission.)

Abstract Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline–deficient (MCD) diet. In these models, Ccl5/ mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Comment Chemokines and their G protein–coupled receptors are increasingly being recognized as crucial mediators in the pathology of chronic disease. Chemokines (chemotactic cytokines) control the movement of immune cells along a concentration gradient to the site of inflammation or tissue injury and are, therefore, intimately associated with the processes involved in wound healing. In chronic liver disease, resident hepatic cells secrete chemokines in response to tissue injury; subsequently, there is additional production by the resulting inflammatory infiltrate, which includes T cells, dendri354

EDITORS Kris Kowdley, Seattle, WA Geoffrey McCaughan, Newtown, Australia Christian Trautwein, Aachen, Germany

tic cells, and macrophages. Hepatic fibrosis is the result of an ongoing wound-healing response to a persistent hepatic insult. The resulting inflammatory response by the liver to this insult leads to the subsequent activation of hepatic stellate cells, which are responsible for the deposition of fibrillar collagens and the development of hepatic fibrosis and cirrhosis. A number of different chemokines, including the CC motif (or CC) chemokines [monocyte chemotaxis protein 1 (MCP-1) or chemokine (C-C motif ) ligand 2 (CCL2); macrophage inflammatory protein 1a (MIP-1a) or CCL3; MIP-1b or CCL4; regulated upon activation, normal T cell expressed, and secreted (RANTES) or CCL5; and eotaxin or CCL11] and the C-X-C motif (or CXC) chemokines [monokine induced by interferon-c or chemokine (C-X-C motif ) ligand 9 (CXCL9) and interferon-inducible protein 10 or CXCL10], have been implicated in the pathogenesis of chronic liver disease.1,2 Likewise, a number of chemokine receptors, including chemokine (C-C motif ) receptor 1 (CCR1), CCR2, CCR5, CCR7, and chemokine (C-X-C motif ) receptor 3, have been shown to play crucial roles in the development of hepatic fibrosis. There is considerable redundancy within chemokine subfamilies,1 with many receptors being capable of binding more than one chemokine and with the same chemokine eliciting a response from more than one receptor (Fig. 1). In a recent study, Berres et al.3 examined the role of the CC chemokine RANTES (also called CCL5) in the interaction between immune cells and hepatic stellate cells and thus in the development of hepatic fibrosis. They examined the expression of RANTES in both human chronic liver diseases (hepatitis C virus and nonalcoholic steatohepatitis) and murine models of hepatic fibrosis, and they demonstrated that T cells in the liver are a major source of RANTES. They then evaluated the effects of the genetic inactivation of RANTES on hepatic fibrosis in animal models of liver disease. Finally, they used the RANTES receptor antagonist Met-CCL5 to assess the effects on both hepatic stellate cell activation in vitro and the development (and treatment) of hepatic fibrosis in animal models of liver injury, and they demonstrated the inhibition of stellate cell activation and the accelerated regression of hepatic fibrosis. This study, therefore, describes the potential

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Fig. 1. CCL/CCR redundancy: human CCRs and their associated ligands. Selected seven-transmembrane–spanning, G protein–coupled CCRs (CCR1-CCR5) and multiple CC chemokines to which they bind are shown. Abbreviations: HCC, hemofiltrate CC chemokine; MDC, macrophagederived chemokine; MPIF-1, myeloid progenitor inhibitory factor 1; TARC, thymus and activation regulated chemokine.

therapeutic utility of blocking the function of RANTES in the treatment of hepatic fibrosis. In this study, Berres et al.3 demonstrated that RANTES was associated with progressive fibrosis in patients with hepatitis C virus, and the distributions of HapMap CCL5 haplotypes were significantly different for patients with mild fibrosis (F0-F1) versus patients with more advanced fibrosis (F2-F4). This difference was principally due to the increased prevalence of the CCL5_H3 haplotype among those with advanced fibrosis (2.6-fold versus those with mild fibrosis). This haplotype is tagged by rs11652536, which is in strong linkage disequilibrium with a functional single-nucleotide polymorphism in the CCL5 promoter that has previously been shown to increase RANTES expression.4 However, this study did not find any significant increases in serum RANTES levels in patients with the minor rs11652536 allele. The involvement of RANTES in progressive fibrosis was also demonstrated in a separate cohort of subjects with nonalcoholic steatohepatitis. The authors suggested that genetically determined serum levels of RANTES may contribute only marginally to increased fibrosis in risk allele carriers. Berres et al.3 then examined the expression of RANTES [messenger RNA (mRNA) and protein] in

two different mouse models of hepatic fibrosis; they used either carbon tetrachloride (CCl4) injections or a methionine and choline–deficient (MCD) diet. Although previous studies have demonstrated elevated expression of RANTES mRNA in animal models of hepatic fibrosis,5 these authors went further by demonstrating that a significant number of RANTESþ cells in the liver were in fact CD3þ T cells. This work also used bone marrow chimeras and Ccl5/ mice to examine the most likely source of RANTES-expressing cells in CCl4-treated mice. RANTES protein expression was markedly reduced (50%-65%) in mice when the bone marrow was transplanted from Ccl5/ mice to wild-type (WT) mice (in comparison with both WT!WT mice and WT!Ccl5/ mice). This experiment showed quite convincingly that hematopoietic cells are likely to be a major source of RANTES associated with hepatic fibrosis, at least in CCl4induced liver injury. The Ccl5/ mice were then used to fully assess the impact of a loss of RANTES expression on the development of hepatic fibrosis in both the CCl4 and MCD models of liver injury. Hepatic fibrosis, which was assessed with Sirius red histochemistry, was markedly suppressed by approximately 65% to 70% in

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both models of liver injury in comparison with WT mice subjected to these fibrotic stimuli. This was confirmed by the significant decrease in hydroxyproline levels and the suppression of transforming growth factor b1, procollagen a1(I), tissue inhibitor of metalloproteinase 1, interleukin-6, and matrix metalloproteinase 9 mRNA expression. There are two additional observations of note from this study. The first is the statistically significant decrease in serum alanine aminotransferase levels observed in Ccl5/ mice as early as 24 hours after a single injection of CCl4. This suggests that in the absence of RANTES expression, there is an early reduction in hepatocyte damage; thus, a role for RANTES-induced inflammatory cells (T cells and macrophages) in hepatocyte damage and/or clearance (as evidenced by the release of alanine aminotransferase) is implied. This is an interesting observation worthy of further investigation. The second observation is the loss of a-smooth muscle actin–positive hepatic stellate cells and myofibroblasts in vivo with RANTES gene inactivation; this is perhaps not unexpected, but the fact that this was not replicated in vitro is interesting because hepatic stellate cells isolated from both WT and Ccl5/ mice and cultured for up to 5 days on plastic showed similar expression levels of a-smooth muscle actin and procollagen a1(I) mRNA. This suggests that hepatic stellate cells require immune cell activation in vivo. In both models, there was a significant reduction in the number of CD3þ T cells and CD68þ macrophages in the livers of Ccl5/ mice versus WT mice. This study clearly demonstrated a requirement for infiltrating immune cells in the development of hepatic fibrosis. This conclusion was confirmed through the use of bone marrow–chimeric mice: CCl5/ bone marrow was transplanted into WT recipients (Ccl5/!WT mice) and vice versa (WT!Ccl5/ mice) after lethal irradiation, with WT!WT mice serving as controls; all mice were subjected to CCl4 injections for 6 weeks. Histological fibrosis, which was assessed with Sirius red histochemistry, was reduced by approximately 75% in the Ccl5/!WT mice, whereas in the WT!Ccl5/ mice, there was a nonsignificant decrease (10%-15%) in hepatic fibrosis versus the WT!WT controls. The final set of experiments in this study used the RANTES receptor antagonist Met-CCL5 in a series of elegantly designed in vitro and in vivo investigations to determine its effect on the activation of hepatic stellate cells (which are known to respond to RANTES) and hepatic fibrosis. Met-CCL5 is a recombinant RANTES analogue that acts as a potent antagonist of the murine

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RANTES receptors CCR1 and CCR5.6,7 Interestingly, Met-CCL5 has no effect on CCR3, a third RANTES receptor7 (Fig. 1). This study showed that Met-CCL5 significantly inhibited the RANTES-induced chemotaxis of hepatic stellate cells and the RANTES-induced secretion of MCP-1 in vitro. Because immune cells were shown in this study to be the principal source of RANTES, the authors incubated hepatic stellate cells with conditioned media from splenocytes isolated from either WT or Ccl5/ mice. They showed a dramatic reduction (35%-45%) in stellate cell chemotaxis, proliferation, and collagen production with Ccl5/ splenocytes. This reduction in fibrogenic activity was even greater when stellate cells were pretreated with Met-CCL5 before the treatment with WT splenocyte–conditioned media (75%-80%). In the in vivo studies, Met-CCL5 (administered concomitantly with either CCl4 or the MCD diet) significantly inhibited hepatic fibrosis progression (20%-40%) and the expression of hepatic genes associated with fibrogenesis. In both animal models of hepatic fibrosis, CD8þ T cells and CD68þ macrophages were significantly reduced by the in vivo MetCCL5 treatment, whereas the numbers of natural killer and natural killer T cells, B220þ B cells, and CD11cþ dendritic cells were unchanged. When daily MetCCL5 treatments were administered after the establishment of fibrosis by an 8-week CCl4 injection regimen (3 days after the final CCl4 injection), they augmented the regression of hepatic fibrosis (50%) after 7 days. These histological changes in fibrosis were preceded by the reduced expression of both procollagen a1(I) and tissue inhibitor of metalloproteinase 1 mRNA levels in the liver. These data are particularly interesting because they suggest the potential for the treatment of established fibrosis via the accelerated regression of fibrotic tissue, although further investigations are warranted to evaluate the mechanisms involved in this process. In a previous study, Ruddell et al.8 identified CD45þ immune cells as a source of RANTES in another murine model of hepatic fibrosis. They used the choline-deficient, ethionine-supplemented dietary model of hepatic injury, liver progenitor cell expansion, and portal fibrosis to demonstrate a role for the tumor necrosis factor family member lymphotoxin b (LTb) in the process of wound healing and hepatic fibrosis.8 They proposed a novel mechanism for RANTES expression by hepatic stellate cells via direct cell contact between liver progenitor and hepatic stellate cells that is induced by the interaction of cell surface–bound LTb on liver progenitor cells with the LTb receptor expressed on hepatic stellate cells. In the same

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study, significant numbers of CD45þ T cells were also demonstrated to express RANTES in choline-deficient, ethionine-supplemented mouse livers and were observed in close spatial association with liver progenitor cells. Neither Ruddell et al. nor Berres et al.3 examined the relative contributions of either T cells or hepatic stellate cells to RANTES expression in these models of hepatic fibrosis. Although it appears that immune cells are the major source of RANTES at least in the CCl4 and MCD models, the contributions of other resident and nonresident hepatic cells require further investigation. Although a number of studies have examined the role of the RANTES receptors CCR1 and CCR5 or RANTES itself in the various processes associated with hepatic fibrosis, Berres et al.3 took a systematic approach in this very comprehensive study to evaluate the role of RANTES; they assessed both the genetic inactivation of the ligand and the antagonistic blockade of the receptors. A similar type of approach was used by Seki et al.,5 who used the genetic inactivation of either CCR1 or CCR5 to examine the effect on hepatic fibrosis in murine models. They demonstrated that the knockout of either of the RANTES receptors had marked inhibitory effects on histological fibrosis. They showed that the profibrogenic effects of CCR1 appeared to be involved in early fibrosis, whereas CCR5 seemed to be principally involved in perpetuating fibrosis. The effects of CCR1 were predominantly mediated by a bone marrow–derived cell population, whereas the profibrogenic effects of CCR5 principally occurred through resident liver cells such as hepatic stellate cells.2 However, as discussed earlier, these chemokine receptors can have multiple additional activation signals from a variety of different ligands, with both MIP-1a and RANTES acting as ligands of both CCR1 and CCR5 (Fig. 1). The inhibitory effects might be attributed to MIP-1a (via CCR1), or MIP1a and/or MIP-1b (via CCR5), just as they were attributed to RANTES by Seki et al. Berres et al. assessed the involvement of RANTES in hepatic fibrosis by using both Ccl5/ mice, and by examining the effects of RANTES receptor antagonism (i.e., via CCR1 and CCR5) with Met-CCL5 and showed very similar effects on the suppression of fibrosis. There are, however, two caveats. Using Ccl5/ mice leaves other CCR1 and CCR5 agonists (Fig. 1) free to activate these receptors and cause infiltration of profibrogenic cells; this may account for the fact that fibrosis inhibition never reached 100% in this study. In addition, Met-CCL5 does not bind CCR3,7 the third RANTES receptor (Fig. 1), and although a few stud-

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ies have examined its role in hepatic fibrosis, the potential exists for RANTES (or even eotaxin), that has been produced as a result of hepatic injury in CCl4- or MCD-treated mice, to exert its profibrogenic effects via this alternate receptor in these models of hepatic fibrosis. Numerous different CCR antagonists that target one of the five different CCRs (CCR1-CCR5) are currently being tested in clinical trials at various stages for the treatment of conditions such as rheumatoid arthritis, asthma, endometriosis, psoriasis, multiple sclerosis, atherosclerosis, chronic obstructive pulmonary disease, cystic fibrosis, and human immunodeficiency virus.9 Previous approaches to the development of chemokine antagonists used neutralizing antibodies for chemokines or their receptors or modified chemokine proteins. Some of these molecules were also found to have limited agonistic properties, which compromised the conclusions drawn in various studies.9 Most compounds in clinical trials are small molecule receptor antagonists; however, neutralizing antibodies remain among those compounds currently being tested, with small peptide–based receptor inhibitors and ribonuclease-resistant RNA aptamers still in preclinical development.9 Chemokine receptor antagonists that block CCR5 have been approved for therapy in patients with human immunodeficiency virus infections. The RANTES receptor antagonist Met-CCL5 has previously been used in numerous in vitro and animal model studies designed to evaluate the role of RANTES in tissue injury and to potentially treat tissue inflammation occurring as a result of cardiac disease, arthritis, bone disease, and lung disease, among other conditions. Some reports have suggested that MetCCL5 is a functional antagonist of CCR5 with partial agonistic activity; this has been evidenced by tyrosine kinase phosphorylation, a small but measurable calcium flux, and a slow internalization of CCR5 in T cells or Chinese hamster ovary K1 cells in vitro.10,11 Others have shown that although Met-CCL5 reduces diet-induced atherosclerosis in animal models,12 RANTES antagonism may not be therapeutically feasible13 because a direct RANTES blockade (as shown in Ccl5/ mice) may compromise systemic immune responses, impede macrophage-mediated clearance of viral infections,14 and impair routine T cell functions.15 Few studies to date have assessed the therapeutic potential of RANTES receptor antagonism on liver disease progression. One such study demonstrated a decrease in liver disease severity in a concanavalin A–induced hepatitis model of T cell–mediated hepatitis in Ccr5/ mice and confirmed the role of

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CCR1þ natural killer cells in the disease process.16 It is apparent that further extensive investigations are required to identify appropriate antagonistic strategies for controlling inflammation and tissue remodeling in clearly defined liver disease contexts. The availability of specific antagonists such as Met-CCL5 will greatly aid us in this endeavor. GRANT A. RAMM, PH.D.

Hepatic Fibrosis Group, Queensland Institute of Medical Research, PO Royal Brisbane and Women’s Hospital, Brisbane, Queensland, Australia

References 1. Karlmark KR, Wasmuth HE, Trautwein C, Tacke F. Chemokinedirected immune cell infiltration in acute and chronic liver disease. Expert Rev Gastroenterol Hepatol 2008;2:233-242. 2. Ramm GA. Chemokine (C-C motif) receptors in fibrogenesis and hepatic regeneration following acute and chronic liver disease. HEPATOLOGY 2009;50:1664-1668. 3. Berres ML, Koenen RR, Rueland A, Zaldivar MM, Heinrichs D, Sahin H, et al. Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice. J Clin Invest 2010;120:4129-4140. 4. An P, Nelson GW, Wang L, Donfield S, Goedert JJ, Phair J, et al. Modulating influence on HIV/AIDS by interacting RANTES gene variants. Proc Natl Acad Sci U S A 2002;99:10002-10007. 5. Seki E, De Minicis S, Gwak GY, Kluwe J, Inokuchi S, Bursill CA, et al. CCR1 and CCR5 promote hepatic fibrosis in mice. J Clin Invest 2009;119:1858-1870. 6. Proudfoot AE, Power CA, Hoogewerf AJ, Montjovent MO, Borlat F, Offord RE, et al. Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J Biol Chem 1996;271:2599-2603. 7. Chvatchko Y, Proudfoot AE, Buser R, Juillard P, Alouani S, KoscoVilbois M, et al. Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3. J Immunol 2003;171:5498-5506. 8. Ruddell RG, Knight B, Tirnitz-Parker JE, Akhurst B, Summerville L, Subramaniam VN, et al. Lymphotoxin-beta receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury. HEPATOLOGY 2009;49:227-239. 9. Anders HJ, Sayyed SA, Vielhauer V. Questions about chemokine and chemokine receptor antagonism in renal inflammation. Nephron Exp Nephrol 2010;114:e33-e38. 10. Wong M, Uddin S, Majchrzak B, Huynh T, Proudfoot AE, Platanias LC, et al. Rantes activates Jak2 and Jak3 to regulate engagement of multiple signaling pathways in T cells. J Biol Chem 2001;276: 11427-11431. 11. Longden J, Cooke EL, Hill SJ. Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells. Br J Pharmacol 2008;153:1513-1527. 12. Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, et al. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ Res 2004;94:253-261. 13. Koenen RR, von Hundelshausen P, Nesmelova IV, Zernecke A, Liehn EA, Sarabi A, et al. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med 2009;15:97-103. 14. Tyner JW, Uchida O, Kajiwara N, Kim EY, Patel AC, O’sullivan MP, et al. CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection. Nat Med 2005;11: 1180-1187.

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15. Makino Y, Cook DN, Smithies O, Hwang OY, Neilson EG, Turka LA, et al. Impaired T cell function in RANTES-deficient mice. Clin Immunol 2002;102:302-309. 16. Ajuebor MN, Wondimu Z, Hogaboam CM, Le T, Proudfoot AE, Swain MG. CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis. Am J Pathol 2007;170:1975-1988. C 2011 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24353 Potential conflict of interest: Nothing to report.

Copper Toxicity in Wilson Disease Explained in a New Way Zischka H, Lichtmannegger J, Schmitt S, Ja¨gemann N, Schulz S, Wartini D, et al. Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease. J Clin Invest 2011;121:1508-1518. (Reprinted with permission.)

Abstract Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b-/- rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper-dependent Fenton chemistry. In a cellfree system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b-/- rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intramitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.

Comment Wilson disease (WD) is an autosomal, recessively inherited copper storage disorder due to mutations of the WD gene ATP7B (adenosine triphosphatase, Cu2þ transporting, beta polypeptide). As a consequence of copper overload, patients develop hepatic and/or neurologic symptoms. Although WD and the causative

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copper overload have been known for decades,1 the molecular pathophysiology of WD is not well understood. Currently, the generally cited mechanism of pathology development in WD involves oxidative damage due to copper overload.2 In this oxidative stress theory, the role of free intracellular copper in initiating generation of reactive oxygen species and consequent oxidative hepatic injury has been proposed. Indeed, several studies in patients with WD and in appropriate animal models indicated that oxidative damage to mitochondria might be involved in hepatic copper toxicity.3,4 However, how can copper cause uncontrolled redox reactions, although there is good evidence that copper is at all times bound to proteins and small molecules and thus is not freely available?5-8 Zischka and colleagues addressed the question whether there might exist an alternative mechanism of how copper overload causes mitochondrial dysfunction in WD and ventured a step beyond current disease concepts. They questioned if oxidative stress is perhaps not the cause, but the consequence of mitochondrial damage in WD. The findings of Zischka and colleagues,9 recently reported in the Journal of Clinical Investigation, indicate that copper overload can directly induce intramitochondrial membrane crosslinking that culminates in mitochondrial destruction and liver failure. With this finding, an important step in the pathogenesis of WD can now be explained in a new way. Zischka and colleagues impressively show in a WD rat model, by use of electron microscopy, that major structural alterations of the mitochondria occur early and parallel to increasing mitochondrial copper content. The alterations clearly precede major functional deficits of the mitochondria and can be reversed by copper-chelating therapy in this early phase. This observation and the fact that signs of oxidative damage were absent in this early phase argues strongly against copper-related oxidative stress as a causative mechanism. In the rat model that was analyzed, clinically evident liver failure occurred late after excessive mitochondrial destruction and subsequent oxidative damage had taken place. After establishing an in vitro cell-free system, the investigators were able to reproduce the observed mitochondrial alterations with isolated control mitochondria exposed to copper under conditions mimicking the physiological intramitochondrial milieu. In this cell-free system, Zischka and colleagues could show that complete mitochondrial destruction occurred only at late stages with massive mitochondrial copper overload and was then paralleled by oxidative damage. As an attempt to explain the observed copper-overload–

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related structural alterations of mitochondria, Zischka and colleagues used a redox proteomics approach and were able to identify three abundant mitochondrial membrane proteins that might form intermolecular thiol bridges between proteins anchored in the outer and the inner mitochondrial membrane under copperoverload conditions. The idea of a copper-enforced intramitochondrial membrane interaction as the underlying mechanism of structural mitochondrial damage is even further supported by the investigators: they demonstrated that the outer membranes of mitochondria of Atp7b/ rats were attached more tightly to the inner mitochondrial membrane compared to controls. Zischka and colleagues draw a convincing line of evidence that in their WD rats and the cell-free system, structural mitochondrial alterations occur at an early stage without any signs of oxidative stress and that these structural alterations might be the result of membrane crosslinking. Yet, are the results of this study applicable to patients with WD? The answer is probably yes, because Sternlieb described a pattern of mitochondrial alterations in hepatocytes of patients with WD that is similar to that observed by Zischka and colleagues in the rat model.10 Sternlieb analyzed hepatocytes of several symptomatic patients with WD and their presymptomatic siblings and could find structural abnormalities in mitochondria in many of the patients, irrespective if they were symptomatic or presymptomatic. Interestingly, in his study, Sternlieb could discriminate three different patterns of mitochondrial damage that seemed to be conserved in each family irrespective of the disease stage. Thus, one can speculate that in patients with WD, the mitochondrial damage processes might differ depending on the underlying disease-causing ATP7B mutation. In addition to explaining the pathophysiology of WD in an innovative way, the results obtained by Zischka and colleagues may even have an impact on current and future therapeutic strategies. In their study, treatment of Atp7b/ animals with copper chelators as well as addition of copper chelators in the cell-free system restored mitochondrial ultrastructure. In addition, the investigators could impressively show that in the Atp7b/ animals, copper-chelator therapy preferentially depleted copper from mitochondria and had only a minor effect on total liver homogenate, liver cytosol, and lysosomes. These results might explain the observation that patients with WD often show good improvement under therapy despite only a marginal decrease in total liver copper content. Future studies will have to clarify if the preferential depletion of

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mitochondrial copper by chelator therapy is also true in patients with WD, and perhaps reflects the central mode of action. In summary, copper, although not freely available, in overload conditions leads to a progressive structural damage of mitochondria via membrane crosslinking. This structural damage is, at least in the animal model, reversible and culminates only in late phases in destruction of the mitochondria with subsequent oxidative stress. Thus, the innovative theory of copperoverload–related mitochondrial membrane crosslinking allows a new view of WD. UTA MERLE, M.D. WOLFGANG STREMMEL, M.D.

Department of Gastroenterology, University of Heidelberg Heidelberg, Germany

References 1. Wilson SAK. Progressive lenticular degeneration: a familiar nervous disease associated with cirrhosis of the liver. Lancet 1912;179: 1115-1119. 2. Burkhead JL, Gray LW, Lutsenko S. Systems biology approach to Wilson’s disease. Biometals 2011; doi:10.1007/s10534-011-9430-9. 3. Mansouri A, Gaou I, Fromenty B, Berson A, Lette´ron P, Degott C, et al. Premature oxidative aging of hepatic mitochondrial DNA in Wilson’s disease. Gastroenterology 1997;113:599-605. 4. Sokol RJ, Twedt D, McKim JM Jr, Devereaux MW, Karrer FM, Kam I, et al. Oxidant injury to hepatic mitochondria in patients with Wilson’s disease and Bedlington terriers with copper toxicosis. Gastroenterology 1994;107:1788-1798. 5. Cobine PA, Ojeda LD, Rigby KM, Winge DR. Yeast contain a nonproteinaceous pool of copper in the mitochondrial matrix. J Biol Chem 2004;279:14447-14455. 6. Cobine PA, Pierrel F, Bestwick ML, Winge DR. Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase. J Biol Chem 2006;281:36552-36559. 7. Lippard SJ. Free copper ions in the cell? Science 1999;284: 748-749. 8. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 1999;284:805-808. 9. Zischka H, Lichtmannegger J, Schmitt S, Ja¨gemann N, Schulz S, Wartini D, et al. Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease. J Clin Invest 2011;121: 1508-1518. 10. Sternlieb I. Fraternal concordance of types of abnormal hepatocellular mitochondria in Wilson’s disease. Hepatology 1992;16:728-732. C 2011 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24405 Potential conflict of interest: Nothing to report.

A Glimpse at the Future of Hepatitis C Therapy: The INFORM Trial Gane EJ, Roberts SK, Stedman CA, Angus PW, Ritchie B, Elston R, et al. Oral combination therapy with a

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nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebocontrolled, dose-escalation trial. Lancet 2010;376: 1467-1475. (Reprinted with permission.)

Abstract Background: Present interferon-based standard of care treatment for chronic hepatitis C virus (HCV) infection is limited by both efficacy and tolerability. We assessed the safety, tolerability, and antiviral activity of an all-oral combination treatment with two experimental anti-HCV drugs—RG7128, a nucleoside polymerase inhibitor; and danoprevir, an NS3/4A protease inhibitor—in patients with chronic HCV infection. Methods: Patients from six centres in New Zealand and Australia who were chronically infected with HCV genotype 1 received up to 13 days oral combination treatment with RG7128 (500 mg or 1000 mg twice daily) and danoprevir (100 mg or 200 mg every 8 h or 600 mg or 900 mg twice daily) or placebo. Eligible patients were sequentially enrolled into one of seven treatment cohorts and were randomly assigned by interactive voice or web response system to either active treatment or placebo. Patients were separately randomly assigned within each cohort with a block size that reflected the number of patients in the cohort and the ratio of treatment to placebo. The random allocation schedule was computer generated. Dose escalation was started in HCV treatment-naive patients; standard of care treatment experienced patients, including previous null responders, were enrolled in higher-dose danoprevir cohorts. Investigators, personnel at the study centre, and patients were masked to treatment allocation. However, the pharmacist who prepared the doses, personnel involved in pharmacokinetic sample analyses, statisticians who prepared data summaries, and the clinical pharmacologists who reviewed the data before deciding to initiate dosing in the next cohort were not masked to treatment allocation. The primary outcome was change in HCV RNA concentration from baseline to day 14 in patients who received 13 days of combination treatment. All patients who completed treatment with the study drugs were included in the analyses. This study is registered with ClinicalTrials.gov, NCT00801255. Findings: 88 patients were randomly assigned to a study drug treatment regimen (n¼74 over seven treatment groups; 73 received at least one dose of study drug) or to placebo (n¼14, all of whom received at least one dose). The median change in HCV RNA concentration from baseline to day 14 ranged from 37 to 52 log(10) IU/mL in the cohorts that received 13 days of combination treatment. At the highest combination doses tested (1000 mg RG7128 and 900 mg danoprevir twice daily), the median change in HCV RNA concentration from baseline to day 14 was 51 log(10) IU/mL (IQR 56 to 47) in treatment-naive patients and 49 log(10) IU/mL in previous standard of care null responders (52 to 45) compared with an increase of 01 log(10) IU/mL in the placebo group. The combination of RG7128 and danoprevir was well tolerated with no treatment-related serious or severe adverse events, no grade 3 or 4 changes in laboratory parameters, and no safety-related treatment discontinuations.

HEPATOLOGY, Vol. 54, No. 1, 2011

Interpretation: This oral combination of a nucleoside analogue polymerase inhibitor and protease inhibitor holds promise as an interferon-free treatment for chronic HCV.

Comment Combination therapy with pegylated interferon (Peg-IFN)/ribavirin (RBV) has been the mainstay therapy of chronic hepatitis C virus (HCV) infection for the last decade.1 However, sustained virological response rates (SVR), which range from 40%-80%, vary considerably with HCV genotypes. Genotype 1 is the most common genotype worldwide and has the lowest SVR rates (40%-50%) with 48 weeks of Peg-IFN/RBV therapy.1 However, not all patients are candidates for PegIFN/RBV therapies, and the multiple side effects associated with this therapy can be a major factor for lack of patient tolerance and treatment discontinuation. Thus, more effective and better tolerated therapies for individuals infected with genotype 1 are needed. Over the past decade, predictors of SVR besides genotype have been identified that have allowed refinement of therapy; these include African American and Hispanic race, coinfection with human immunodeficiency virus, insulin resistance, viral level, and the recently identified interleukin-28B (IL-28) polymorphism. In addition to the above factors, the concept of response-guided therapy, whereby treatment duration is based on the rate of viral clearance from the serum during treatment, has allowed tailoring of therapy, allowing shorter duration of therapy in those who clear HCV RNA rapidly at week 4 (rapid virological response). Identification of the structural and nonstructural (NS) proteins of the hepatitis C genome has led to identification of targets to directly inhibit viral replication. The NS3/4A is a serine protease (NS3) and cofactor (NS4A) that catalyzes the posttranslational processing of NS proteins from the polyprotein that is essential for viral replication.2 The NS3 protease cleaves NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions.2 The products released go on to form a replicative complex responsible for forming viral RNA. Thus, NS3/4A provides an ideal target for antiviral therapy.3,4 The HCV NS5B RNA-dependent RNA polymerase is a key enzyme involved in HCV replication, catalyzing the synthesis of the complementary minus-strand RNA and subsequent genomic plus-strand RNA from the minus-strand template and is also an ideal target for inhibiting HCV replication. Direct-acting antiviral agents (DAAs) target the HCV-encoded proteins and when added to Peg-IFN/RBV have resulted in improved SVR rates compared to standard of care.5,6

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Telaprevir and boceprevir are two NS3/4a protease inhibitors for which phase 3 trials are completed. In these trials,5-7 telaprevir and boceprevir are both added to Peg-IFN/RBV, with substantial improvement in SVR rates in both treatment-naive patients and prior nonresponders. These agents are associated with additional side effects including anemia, skin rash, and gastrointestinal symptoms. Both telaprevir and boceprevir have been shown to cause rapid selection of resistanceassociated variants when given as monotherapy, and neither DAA should be administered without PegIFN/RBV.8 Studies with both drugs have shown that optimal doses of RBV are needed to maximize SVR rates and minimize the development of resistance-associated variants. The resistance profile of triple therapy with boceprevir is similar to that of telaprevir in patients who fail to achieve SVR,5 and cross-resistance against other NS3 protease inhibitors may occur.8,9 These resistant strains have been found to persist after withdrawal of therapy with telaprevir and boceprevir in combination with Peg-IFN/RBV and can persist up to 3 years.8,9 The INFORM-1 (Interferon-Free regimen for the Management of HCV) trial is the first randomized, double blind, placebo-controlled, dose escalation trial performed in six centers in New Zealand and Australia. This trial was designed to examine the safety of two new direct-acting antiviral drugs: RG7128 and danoprevir. RG7128 is a 30 50 -di-isobutyric acid ester prodrug of the cytosine nucleoside analogue b-D-20 deoxy-20 -fluoro-20 C-methylcytidine. This compound’s triphosphate form inhibits HCV NS5b RNA polymerase. Danoprevir is a macrocyclic inhibitor of HCV NS3/4A protease, which differs from the linear protease inhibitors telaprevir and boceprevir. The addition of RG7128 to danoprevir is an important milestone as the combination of DAAs in the treatment of hepatitis C has the potential to reduce the emergence of resistant associated variants. Moreover, therapies that can be effective in patients with hepatitis C genotype 1 infection without Peg-IFN/RBV will make treatment possible for the many patients who have contraindications to Peg-IFN therapy. Eighty-eight genotype 1–infected Caucasian patients without cirrhosis who had a minimum HCV RNA of 105 IU/mL were randomized in the INFORM-1 trial, including both treatment-naı¨ve (n ¼ 66) and treatment-experienced patients (n ¼ 22). The group of treatment-experienced patients included those with prior relapse, non-null responders, or null responders (less than 1 log10 IU/mL reduction in HCV RNA concentration after 1 month, or less than 2 log10 IU/mL

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HEPATOLOGY, July 2011

Fig. 1. Week 13 viral response rates for INFORM-1 cohorts F and G.

reduction after 12 weeks). Patients were randomized into nine different treatment groups and placebo, where all groups received therapy with danoprevir and RG7128 in ascending dose combinations for 7-13 days followed by standard therapy with RBV and Peg-IFN, with the highest doses tested being RG7128 at 1000 mg twice daily and danoprevir at 900 mg twice daily. The primary outcome in this study was the change in HCV RNA concentration from baseline to day 14 in patients who received 13 days of combination treatment. In the highest dose cohorts, five of eight treatmentnaive patients and two of eight null responders had HCV RNA concentrations below the limit of detection (