Summary - Edinburgh Research Explorer



Antifibrotic therapies in chronic liver disease: tractable targets and translational challenges Prakash Ramachandran and Neil C Henderson*Dr Prakash Ramachandran (MBChB, PhD)MRC Centre for Inflammation Research,Queen's Medical Research Institute,University of Edinburgh,47 Little France Crescent,Edinburgh,Scotland, UKEH16 4TJ*Corresponding AuthorProfessor Neil C Henderson (MBChB, PhD)MRC Centre for Inflammation Research,Queen's Medical Research Institute,University of Edinburgh,47 Little France Crescent,Edinburgh,Scotland, UKEH16 4TJTel: +0044 131 242 6653E-mail: Neil.Henderson@ed.ac.ukSummaryChronic liver disease prevalence is increasing globally. Iterative liver damage, secondary to any cause of liver injury, results in progressive fibrosis, disrupted hepatic architecture, and aberrant regeneration, which are defining characteristics of liver cirrhosis. Liver transplantation is an effective treatment for end-stage liver disease, however demand greatly outstrips donor organ supply, and in many parts of the world liver transplantation is unavailable. Therefore, effective antifibrotic therapies are urgently required. In this review we discuss the rapid progress that has been made in the identification of potentially tractable cellular and molecular antifibrotic targets, and describe some of the completed and ongoing clinical trials of antifibrotic agents in patients with chronic liver disease. We then examine where the main translational challenges lie, in terms of successful conversion of scientific discoveries into potent antifibrotics, and the strategies that are currently being employed to facilitate successful bridging of the ‘translational gap’ between putative therapeutic targets and effective therapies for patients with chronic liver disease. Chronic liver disease secondary to virtually any aetiology results in extracellular matrix (ECM) deposition and scar formation, known collectively as liver fibrosis. Iterative liver injury, usually over many years, results in progressive fibrosis and ultimately leads to disrupted hepatic architecture, vascular changes, and aberrant regeneration, which are defining characteristics of liver cirrhosis. Currently, treatment options for patients with chronic liver disease are limited to removal of the underlying cause, if possible, and management of the complications of liver cirrhosis, with liver transplantation reserved for a select few. However, with the incidence of cirrhosis increasing, the paucity of donor organs available, and liver transplantation unavailable in many parts of the world, potent antifibrotic therapies are urgently required. Importantly, the degree of liver fibrosis predicts adverse clinical events including clinically significant portal hypertension1, 2, hepatic decompensation3 and the development of hepatocellular carcinoma (HCC)4. Therefore, developing effective antifibrotic therapies for patients with chronic liver disease is likely to impact significantly on morbidity and mortality, and hence, the discovery of effective antifibrotic therapies remains the holy grail in hepatology. In this review we discuss some of the key cellular and molecular mechanisms driving hepatic fibrosis, the recent progress on the identification of tractable antifibrotic targets, the current antifibrotic clinical trials in patients with chronic liver disease, and the hurdles that still need to be overcome to allow successful bridging of the ‘translational gap’ between putative therapeutic targets and effective antifibrotic therapies.Liver fibrosis is dynamic and potentially reversibleLong thought to be relentlessly progressive and irreversible, it is now clear in both pre-clinical animal models and human liver disease that liver fibrosis is a highly dynamic process, with the potential for regression as well as progression5. Such reversibility has been demonstrated in virtually all aetiologies of chronic liver disease following removal of the causative agent. The data is most compelling in patients with chronic viral hepatitis where large-scale clinical trials6-8, with paired liver biopsies pre- and post-antiviral therapy, have convincingly shown that even patients with cirrhosis can show fibrosis regression. Importantly, improvements in liver fibrosis are associated with better clinical outcomes, with “regressors” having higher 10-year survival than “non-regressors” following successful treatment for chronic hepatitis C infection (HCV)9. Hence, treatments to improve hepatic fibrosis are both feasible and potentially highly beneficial, even in those with significant fibrotic liver disease.However, despite effectively removing the pro-fibrogenic stimulus in the liver by curing HCV infection, only a minority of cirrhotic patients become non-cirrhotic (as assessed by non-invasive means)10, whilst portal hypertension may even progress following virological cure11 and there remains a persistent risk of developing HCC10. Thus, treating the causative agent is not in itself sufficient to reliably improve clinical outcomes in all patients with liver fibrosis, again highlighting the urgent need for additional directly-acting potent antifibrotic therapies.Cellular targets in hepatic fibrosisEpithelial cell damageA key feature of fibrotic diseases, not only in the liver but also in other organs, is the presence of epithelial cell injury as an initiator and driver of progressive scar deposition12. Within the liver, epithelial cell damage can affect either biliary epithelium (as in the case of biliary fibrotic disorders such as primary sclerosing cholangitis (PSC) or primary biliary cirrhosis (PBC)), or hepatocytes (as in the case of other causes of chronic liver disease such as alcohol-related, viral hepatitis, non-alcoholic fatty liver disease or metabolic liver diseases). Theoretically, if epithelial injury can be abrogated, then this may prevent fibrosis progression and indeed potentially allow other cell types to facilitate scar resolution. However, as described above, even when the injurious agent is entirely removed, such as achieving sustained virological response (SVR) in the treatment of HCV, this is not always sufficient to have a clinically meaningful impact on cirrhosis and the risk of long-term complications. This may reflect the fact that by the time some hepatic fibrosis patients present clinically, the “wound-healing” response has become aberrant, self-perpetuating and independent of ongoing epithelial damage, which may have already “burnt out”. Nevertheless, multiple approaches to directly target epithelial damage as an antifibrotic strategy are currently being assessed. Caspase inhibitors, which are thought to inhibit epithelial cell apoptosis, have been effective in animal models of non-alcoholic fatty liver disease (NAFLD) and are undergoing assessment in clinical trials in a broad variety of liver disease settings13, 14. Similarly cathepsin B inhibition, which blocks a lysosomal apoptosis pathway, reduces liver injury and fibrosis in animal models of biliary injury15. However, given that cirrhosis predisposes to HCC formation, caution will need to be exercised when using therapies to promote epithelial cell survival in the context of fibrotic liver disease.Hepatic myofibroblastsMyofibroblast accumulation is a feature of fibrosis in virtually all organs including the liver16, 17 , and myofibroblasts are the major source of pathological ECM during hepatic fibrogenesis. The precise origin of myofibroblasts in both the liver and other organs remains an area of active investigation. Within the fibrotic liver the principal scar-producing cells are activated hepatic stellate cells (HSCs)18, 19, whilst a distinct population of portal myofibroblasts may predominate in biliary fibrosis20, 21. HSCs are pericytes which reside within the space of Disse between hepatocytes and endothelial cells and encircle the liver sinusoid. In response to epithelial injury, HSCs transdifferentiate and proliferate to generate activated myofibroblasts. In addition to the production of pathological ECM, this population has a number of additional pro-fibrogenic properties, including immunomodulatory functions22 and secretion of proteins such as tissue inhibitor of metalloproteinase 1 (TIMP-1)23 which inhibit fibrosis resolution. Given that hepatic myofibroblasts are the major source of ECM during liver fibrosis, they represent a key target in antifibrotic therapy development. Firstly, by understanding the signals promoting HSC activation to a myofibroblast phenotype, treatments could be developed to inhibit myofibroblast formation and consequently prevent fibrogenesis. Perhaps unsurprisingly, these activating signals are myriad and complex, and are reviewed in depth elsewhere24, 25. However, this burgeoning literature has highlighted a number of potential antifibrotic therapeutic targets25-27 (Table 1). These data have stimulated several antifibrotic clinical trials, aimed at inhibiting myofibroblast activation26, 27 (Table 2) Table 1: Summary of signals promoting hepatic stellate cell activation in liver fibrosis. Adapted from Lee et al.25 and Yoon et al.26. Potential antifibrotic targetsCytokines/ChemokinesTGF-βPDGF CTGF/CCN2Pro-inflammatory cytokines (e.g. TNF-α, IL-1β)CCR2CCR5Membrane ReceptorsCannabinoid Receptor (CB1)Angiotensin II type 1 (AT1) receptorSerotonin (5-HT2B receptor)Endothelin-1Nuclear ReceptorsPeroxisome-proliferator activator receptors (PPAR) –α, -δ and-γVitamin D ReceptorTyrosine KinasesDownstream signalling of range of growth factors AdipokinesLeptinAdiponectinGhrelinPattern Recognition ReceptorsTLR activation by intestinal dysbiosisInteractions with ECMIncreased matrix stiffnessECM-induced integrin signallingAutophagyAutophagy proteinsReactive Oxygen SpeciesNADPH Oxidases (NOX1, NOX2, NOX4)Epigenetic changesDNA Methylation via DNA methyl transferases or MeCP2Histone modifications by HDAC enzymes or myocardin-related transcription factor A (MRTF-A)Micro-RNAs (e.g. miR-21)Abbreviations: CCR, chemokine receptor; TGF-β, transforming growth factor-β; PDGF, Platelet-derived growth factor; CTGF/CCN2, Connective tissue growth factor; TLR, Toll-like receptor; NAFLD, Non-alcoholic fatty liver disease; ECM, extracellular matrix; MeCP2, methyl-CpG binding protein 2; HDAC, histone deacetylase. An alternative strategy to targeting the hepatic myofibroblast activation process would be to promote myofibroblast removal from the fibrotic liver. During fibrosis resolution, following the cessation of injury, there is a dramatic loss of the activated myofibroblast population by apoptosis28, 29, senescence30 or reversion to a quiescent phenotype31, 32 (Fig 1). Apoptosis is mediated by a loss of pro-survival signals such as interleukin-1β (IL-1β)33, tumour necrosis factor- α (TNF-α)33, TIMP-134 and integrin signalling35 as well as the direct action of pro-apoptotic molecules including nerve growth factor (NGF)36, adiponectin, and TNF-related apoptosis-inducing ligand (TRAIL)24. However, attempts to therapeutically target such ubiquitous pro-apoptotic pathways systemically are likely to be limited by off-target toxicity. An effective strategy may therefore be to develop methods to deliver treatments directly to the pro-fibrogenic cell population. For example, gliotoxin, a fungal product, can induce cellular apoptosis and can be targeted to activated hepatic myofibroblasts by conjugation to a specific antibody domain, with consequent antifibrotic effects37. Alternatively, Vitamin A-coupled liposomes can deliver siRNA to heat-shock protein 47 (HSP47) specifically to HSCs, promoting myofibroblast apoptosis and inhibiting liver fibrosis38. This HSP47 targeting nanoparticle (ND-L02-s0201) is currently undergoing clinical trials. Myofibroblast senescence, following the cessation of liver injury, may result in a less fibrogenic phenotype and facilitate their removal by natural killer (NK) cells30. The signals promoting myofibroblast senescence include CCN1/CYR61 (cysteine rich protein 61)39, 40 and IL-2241, which consequently have an antifibrotic effect in preclinical models of chronic liver disease. Interestingly, atorvastatin can also induce myofibroblast senescence42, which would be consistent with retrospective clinical studies in patients with chronic hepatitis B43 or hepatitis C infection44, 45, which suggest that statin use is associated with slower progression of liver fibrosis. Whilst such studies have limitations in terms of potential confounding variables and the consistency of statin dose/type, at the very least they demonstrate that statins are safe in chronic liver disease and that the antifibrotic activity of statins should be further assessed in prospective studies. Finally, activated HSCs can exhibit plasticity, with reversion back to a quiescent HSC phenotype, although the reverted HSCs seem “primed” to reactivate in response to further fibrogenic stimuli31, 32. Recently, two studies have harnessed the plasticity of hepatic myofibroblasts by directly reprogramming these cells into hepatocyte-like cells during liver fibrosis46, 47. Song et al. demonstrated that in?vivo expression of four transcription factors (FOXA3, GATA4, HNF1A, and HNF4A) from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis46. Furthermore, Rezvani et al. have shown in lineage-tracing mice that adeno-associated virus 6 (AAV6) vector-mediated in vivo hepatic reprogramming of liver myofibroblasts (using AAV6 expressing the transcription factors FOXA1, FOXA2, FOXA3, GATA4, HNF1A, and HNF4A) generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis47. The ability to specifically target hepatic myofibroblasts within the liver and reprogramme them into cells with a positive functional benefit has great therapeutic potential, however further research and development in this area will clearly be required to assess both the safety and efficacy of viral vector-based cellular reprogramming approaches in patients with hepatic fibrosis. The relative contribution of HSC/myofibroblast apoptosis, senescence and plasticity in human liver disease remains to be elucidated, but enhanced understanding of the processes which govern the fate of these pro-fibrogenic cells will hopefully yield effective antifibrotic therapies.Liver macrophagesIn addition to epithelial cell injury and myofibroblast accumulation, liver fibrosis is also characterised by a complex multicellular immune response. In particular, cells of the monocyte/macrophage lineage are temporally and spatially associated with liver myofibroblasts and scar tissue48-50. A series of functional studies have demonstrated that during ongoing liver injury, macrophages can be pro-fibrogenic5, at least in part via the expression of mediators such as transforming growth factor-β (TGF-β)51, TNF-α and IL-1β33, which promote myofibroblast activation and survival. Conversely, macrophages are also critical for the resolution of liver fibrosis48, 50, 52, producing scar-degrading matrix metalloproteinase (MMP) enzymes49, 50, 53 and molecules such as TRAIL54 which promote myofibroblast apoptosis. This dichotomous function of liver macrophages is largely explained by the significant heterogeneity in macrophage populations within the liver. Analysis of murine macrophage subpopulations have identified that following liver injury there is recruitment of a subpopulation of circulating monocytes (Ly-6Chi cells) via the CCR2/CCL2 chemokine axis51, 55, 56, which act in a pro-fibrogenic manner. These pro-fibrogenic Ly-6Chi macrophages then undergo a change in phenotype to form a “restorative” Ly-6Clo hepatic macrophage population, which is critical for fibrosis resolution50. These Ly-6Clo cells downregulate the expression of pro-inflammatory cytokines and chemokines, whilst increasing the expression of MMPs and other antifibrotic genes such as CX3CR157 and arginase-158. A similar monocyte/macrophage infiltration is seen in cirrhotic human liver59, 60, with the capacity for phenotypic switching also observed59. However, the specific mediators produced by distinct human hepatic macrophage subpopulations remains to be elucidated. Given their key role in both fibrogenesis and fibrosis resolution, macrophages represent an attractive target for antifibrotic therapies. Inhibition of monocyte recruitment using CCR2-deficient mice resulted in less myofibroblast activation and reduced liver fibrosis in response to chronic injury51, 55. Similarly, blockade of vascular endothelial growth factor (VEGF) reduces liver sinusoidal permeability and monocyte recruitment with a consequent antifibrotic effect52. However, inhibition of monocyte recruitment in these preclinical studies also reduced fibrosis resolution52, 55, suggesting a more nuanced therapeutic approach will be required to effectively target differing subpopulations of macrophages in the context of hepatic fibrosis. A possible strategy may involve the development of methods to manipulate the hepatic macrophage phenotype in vivo, thus modifying the balance of pro- and anti-fibrotic macrophage populations. Proof of this concept has been demonstrated in murine models using a Spiegelmer technique (short L-enantiomeric RNA molecules which specifically inhibit proteins of interest) to inhibit CCL261 or by administering liposomes50, both of which enhance pro-resolution macrophages and accelerate fibrosis regression. Whether such methods will be effective in human fibrotic liver disease remains to be seen.Other immune cellsIn addition to macrophages, the fibrotic liver is characterised by infiltration of numerous other immune cell populations. In order to try to dissect and understand the immune cell network during liver fibrosis many studies have taken a reductionist approach, focussing on the role of specific immune cell populations during both hepatic fibrogenesis and fibrosis resolution62.NK cells: This population have been shown to have antifibrotic effects, by killing of activated HSCs due to expression of IFN-γ63 or death receptors/ligands64, or by the clearance of senescent myofibroblasts30. Enhancing NK cell activity by inducing hepatic IFN-γ promotes fibrosis resolution in murine models30.Neutrophils: A major regulatory role for neutrophils in liver fibrosis has not been clearly demonstrated as yet65. Neutrophils express MMPs which can promote fibrosis resolution66, although it remains uncertain whether neutrophils are present in sufficient numbers in the human cirrhotic liver to modulate liver fibrosis resolution.Dendritic cells (DCs): Hepatic DCs may promote ECM degradation via MMP9 expression67, and can be expanded by administering Flt3L (fms-like tyrosine kinase-3 ligand) leading to antifibrotic effects67.T cells: Similar to macrophages, T cells can potentially have pro- or anti-fibrotic roles in the diseased liver. Specifically, TH2 responses have been shown to be profibrogenic68 whilst the presence of TH1 responses68 or regulatory T cells (Treg)69 result in less fibrosis. However, as with many aspects of the cellular biology regulating liver fibrosis, this is likely to be an overly simplistic view with other populations such as TH17 cells70 or cytotoxic T cells co-existing, whose function in liver fibrosis has yet to be fully defined62. An example of where targeting of T cell biology may prove to be a fruitful therapeutic avenue in the near future is in PSC, a condition where gut-homing CCR9+ effector T cells are aberrantly recruited to the liver and may promote biliary damage, and can potentially be blocked using existing agents such as the integrin α4β7 blocker Vedolizumab or the CCR9 chemokine receptor inhibitor CCX282B71.Liver sinusoidal endothelial cells (LSECs)LSECs are highly specialised endothelial cells which when differentiated have the capacity to promote HSC quiescence and modulate hepatic immune responses. However, following liver injury, and preceding fibrosis, LSECs de-differentiate (a process known as capillarization), which promotes HSC activation and fibrogenesis72. The exact mediators which control this cellular interaction are yet to be fully defined, but the use of therapies such as soluble guanylate cyclase (sGC) activators to promote LSEC differentiation have been shown to inhibit fibrosis and promote fibrosis resolution in animal models72. Furthermore, chemokine receptor expression is critical in defining LSEC function, with loss of CXCR7 and upregulation of CXCR4 expression promoting a pro-fibrogenic LSEC phenotype, whilst administration of a CXCR7 agonist was antifibrotic73. Similarly CXCL9, a ligand for CXCR3, inhibits the capacity of LSECs to activate hepatic myofibroblasts and is antifibrotic in animal models of liver disease74. Hence, manipulation of chemokine pathways may enable modulation of LSEC phenotype to inhibit fibrosis.Angiogenesis, the formation of new blood vessels from existing ones, is a key process during liver fibrogenesis and the development of portal hypertension75. LSEC proliferation and migration are critical for hepatic angiogenesis, as part of a multicellular response involving hepatocytes, hepatic myofibroblasts and immune cells which results in the release of pro-angiogenic mediators such as VEGF and angiopoietin75, 76. Ultimately, these changes result in the characteristic angioarchitectural abnormalities seen in cirrhosis. Anti-angiogenic therapies such as VEGF blocking antibodies or tyrosine kinase inhibitors (e.g. Sorafenib or Sunitinib), have therefore been tested in pre-clinical models of liver fibrosis and in general have antifibrotic effects77. Given that these agents are already in clinical use in oncology, it would be appealing to trial them as antifibrotics. However, recent data have suggested that VEGF signalling52 and angiogenesis78 may also have a role in liver fibrosis resolution, so global blockade could also potentially have deleterious effects.The rise of core pathways in liver fibrosisIn addition to the cellular targets for liver antifibrotic therapies described above (Fig 2), it is appealing to develop therapeutic strategies to manipulate key fibrosis pathways. However, given the complexity of the cellular and molecular interactions which drive fibrosis, the concept of “core” and “regulatory” pathways has come to the fore as a potential means to identify antifibrotic drug targets which might be relevant in multiple different organ fibroses, and therefore more readily translated79. “Core” pathways are those which are essential for fibrosis and are conserved across organs and species, whilst “regulatory” pathways will still have a substantial role in fibrosis but may vary between organs, species and individuals. By targeting core fibrosis pathways, treatments can potentially be utilised in multiple organs and diseases, accelerating the pipeline to effective translation. For example, in idiopathic pulmonary fibrosis (IPF), rapidly progressive fibrosis and high mortality rates (median survival of patients is only two to three years) have led to clinical trials and subsequent approval of antifibrotic therapies such as pirfenidone80 and nintedanib81, which are now being tested in chronic liver disease. However, the ubiquitous nature of core pathways suggests important roles in maintaining tissue homeostasis in non-fibrotic organs, so caution will need to be exercised when therapeutically manipulating these pathways to minimise off-target effects. Lysyl oxidase-like-2 (LOXL2)Lysyl oxidase-like-2 (LOXL2) is a matrix enzyme responsible for crosslinking of collagen fibrils and hence rendering scar tissue more resistant to degradation. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023), as well as being efficacious in both primary and metastatic xenograft models of cancer, reduced fibrosis in models of liver and lung fibrosis. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, endothelial cells and desmoplasia , with decreased production of growth factors and cytokines, and attenuated TGF-β pathway signaling82. Clinical trials of a monoclonal antibody targeting LOXL2 in liver fibrosis are now underway.TGF-β and v integrinsThe TGF-β pathway is a classic example of a core pathway in fibrosis, as TGF-β is arguably the most pro-fibrogenic cytokine known, and has been shown to drive fibrosis in multiple organs and disease states83. However, TGF-β has numerous other regulatory roles in both health and disease, from immune regulation to carcinogenesis, so global blockade of this pathway is unlikely to be feasible in a clinical setting. Therefore, strategies to selectively interfere with the TGF-β pathway in the context of fibrosis are required. Importantly, in order to mediate its pro-fibrogenic effects, TGF-β requires activation from a latent form (whilst bound in the ECM) to an active form, and v integrins have been shown to be major regulators of the TGF-β activation process. Furthermore, recent pre-clinical studies in the liver and other organs, have shown that inhibition of various members of the αv integrin family reduces fibrosis in multiple organs and disease states, highlighting αv integrin-mediated TGF-β activation as a core, targetable pathway in fibrosis84-86.Antifibrotic clinical trials in chronic liver diseaseAs discussed above, there are now a plethora of cellular and molecular targets for antifibrotic therapies in chronic liver disease. This has led to a number of clinical trials with liver fibrosis as a primary or secondary endpoint. Indeed, a search on for liver or hepatic interventional studies with fibrosis as an outcome, yields over 240 trials. Relevant antifibrotic studies are summarised in Table 2, having excluded those where treatments for specific causes of liver disease are being assessed (e.g. antiviral drugs in chronic viral hepatitis, immunosuppression in autoimmune liver disease, weight loss in NAFLD) or where the mechanism of action of the study compound is unclear. Many of these studies are still ongoing, although so far a consistently proven antifibrotic therapy in liver disease has not been identified. The studies that do suggest a possible antifibrotic effect for a specific treatment have thus far been in small numbers of patients and have not been replicated in larger studies as yet. This highlights the need for a more effective and consistent translational strategy in this area in the coming years.Table 2: Clinical trials of potential antifibrotic therapies in chronic liver disease. Summary of clinical trials where changes in liver fibrosis are listed as primary or secondary outcome. Interventional studies identified from and table adapted from Schuppan et al.87 and Yoon et al.26. Trials where treatments for specific causes of liver disease are being assessed (e.g. antiviral therapy) have been excluded. TreatmentBiological targetAetiology of Liver DiseaseNumber of PatientsTrial OutcomeReference or NumberReduce Epithelial InjuryGS-9450Pan-caspase inhibitorHCV307Study terminated00874796EmricasanPan-caspase inhibitorNAFLD330Ongoing02686762Post-transplant HCV60Results pending02138253MetforminReduce insulin resistanceNAFLD80Results pending00134303NAFLD110Reduced fibrosis88NAFLD86Results pending02234440LiraglutideGLP-1 agonistNAFLD52Reduced fibrosis progression89ExenatideGLP-1 agonistNAFLD20Study terminated00529204NAFLD60Results pending01208649OltiprazAMPK activator which reduces insulin resistanceNAFLD83No effect on fibrosis90NAFLD283Results pending02068339MSDC-0602KInsulin sensitiserNAFLD283Results pending02068339Ethyl-eicosapentanoic acidAnti-oxidantNAFLD243No effect on fibrosis91MetadoxineAnti-oxidantNAFLD108Ongoing02541045Omega-3 fish oilsAnti-oxidantNAFLD41No effect on fibrosis92NAFLD100Results pending00760513Obeticholic acidFarnesoid receptor X (FXR) agonistNAFLD283Reduced fibrosis 93NAFLD2000Ongoing02548351PBC217Results pending01473524PBC350Ongoing02308111PSC75Ongoing02177136VolixibatApical Sodium-Dependent Bile Acid Transporter Inhibitor (ASBTi)NAFLD266Ongoing02787304AramcholFatty acid-bile acid conjugateNAFLD240Ongoing02279524MetreleptinLeptin analogueNAFLD20Results pending01679197Inhibit Hepatic Myofibroblast Activation or Promote Myofibroblast Loss LosartanIrbesartanCandesatanAngiotensin II type 1 (AT1) receptor inhibitorsNAFLD214Results pending01051219HCV14No effect on fibrosis94HCV166Results pending00265642ALD85Reduced fibrosis95AtorvastatinHMG-CoA reductase inhibitorNAFLD150Not reported01987310PioglitazoneRosiglitazoneFarglitazarPeroxisome-proliferator activator receptor (PPAR) –γ agonistHCV209No effect on fibrosis96HCV/HIV31Not reported00742326NAFLD55No effect on fibrosis97NAFLD74Reduced fibrosis progression98NAFLD53No effect on fibrosis99NAFLD247No effect on fibrosis100NAFLD90Ongoing01068444ElafibrinorPeroxisome-proliferator activator receptors (PPAR) –α and –δ agonistNAFLD276Reduced fibrosis in elafibrinor responders101NAFLD2000Ongoing02704403BenzafibratePPAR –α, –γ and –δ agonistPBC100Results pending01654731Vitamin DVitamin D ReceptorNAFLD200Results pending01623024NAFLD60Results pending01571063HBV1500Ongoing02779465Anti-CTGF monoclonal antibodyCTGF/CCN2 blockadeHBV228Results pending01217632GS-4997 +/- SimtuzumabASK1 inhibitor +/- Anti-LOXL2 mAbNAFLD72Results pending02466516PRI-724Wnt signalling inhibitorHCV18Pending02195440ND-L02-s0201Vitamin A-coupled liposome targeting HSP47Moderate to extensive fibrosis25Pending02227459RimonabantCB1 receptor agonistNAFLD165Study terminated00576667NAFLD89Study terminated00577148Modulate Immune ResponsesPentoxyfyllineTNF-α (pro-inflammatory cytokine) inhibitionNAFLD55Possible reduction in fibrosis 102HCV100Not reported00119119IL-10Anti-inflammatory cytokineHCV30Reduced fibrosis103Interferon-γImmunomodulationHCV502No effect on fibrosis104HCV20No effect on fibrosis105HBV99Reduced fibrosis106PF-4136309CCR2 inhibitorHCV24Study terminated01226797MaravirocCCR5 inhibitionHIV + coinfection with HBV and/or HCV138Results pending01327547CenicrivrocCCR2/CCR5 inhibitionNAFLD289Results pending02217475ViusidNutritional supplement (anti-inflammatory and anti-oxidant)HCV100Reduced fibrosis107Target Core Fibrosis PathwaysPirfenidoneTGF-β inhibition (along with anti-inflammatory effects and inhibition of HSP47)HCV150Results pending02161952HydronidonePirfenidone derivativeHBV240Ongoing02499562SimtuzumabAnti-LOXL2 mAbHCV +/-HIV18No effect on fibrosis108PSC235Results pending01672853Abbreviations: CCR, chemokine receptor; TGF-β, transforming growth factor-β; CTGF/CCN2, Connective tissue growth factor; NAFLD, Non-alcoholic fatty liver disease; HCV, hepatitis C virus; HBV, hepatitis B virus; ALD, alcohol-related liver disease; ASK-1, apoptosis-signal regulation kinase 1; GLP-1, Glucagon-like peptide 1; HBV, hepatitis B virus; AMPK, adenosine monophosphate-activated protein kinase; PBC, primary biliary cirrhosis; PSC, Primary sclerosing cholangitis; LOXL2, lysyl oxidase-like-2; HSP47, Heat shock protein-47; CB1, Cannabinoid recptor-1. Translational challenges in liver fibrosis‘Humanising’ liver fibrosis researchDespite numerous cellular and molecular targets and a large number of clinical trials, there are still no antifibrotic therapies licensed for the treatment of patients with chronic liver disease. There are multiple reasons for this, as chronic liver disease poses a number of unique challenges to the translational pipeline (Fig 3). Principally, the vast majority of therapeutic targets have been identified in preclinical animal models of liver fibrosis. Whilst there are undoubted similarities between these models and human disease in terms of histology, cellular dynamics and molecular changes, the fact remains that human cirrhosis usually develops over decades in contrast to rodent models where fibrosis is induced over weeks or months, and the degree of fibrosis is almost invariably significantly less than that seen in human cirrhosis. In addition, human fibrotic liver disease displays a more complex ECM (including collagen cross-linking and elastin deposition) with prominent vascular changes109, important alterations that are often not recapitulated by animal models. Hence, when using pre-clinical rodent models of liver fibrosis to identify antifibrotic targets, many research groups interrogate the putative therapeutic target using multiple models of hepatic fibrosis, with varied modes of injury. As discussed above, fibrosis resolution is well described in human liver disease5. However, liver fibrosis resolution is not well characterised in all rodent models. Thus, when investigating antifibrotic treatments which could promote fibrosis regression, researchers need to either provide a detailed characterisation of fibrosis regression in their model or utilise models such as carbon tetrachloride (CCl4)50, bile duct ligation52 or methionine-choline deficient (MCD) diet61, where both fibrosis progression and resolution have been reliably demonstrated.It is also increasingly clear that we should no longer view the translational pipeline as a simple ‘one-way’ street, linearly progressing from cell-culture based assays to rodent pre-clinical models to attempts at clinical translation, as this model has largely failed to deliver potent antifibrotic therapies both in the context of chronic liver disease and other organ fibroses. Instead, we should consider the translational pipeline as bi-directional, with the data accrued from in-depth cellular and molecular analysis of human fibrotic livers significantly shaping and guiding the pre-clinical evaluation of potential therapeutic targets. Furthermore, “omics” technology now allows us to interrogate human fibrotic liver tissue on an unprecedented scale, from whole tissue all the way down to the level of single cells. This provides huge opportunities to delineate, with ever-increasing resolution, the key cellular and molecular pathways which regulate human liver fibrosis, across the whole gamut of chronic liver disease. Finally, the use of novel experimental techniques such as “humanised” mouse models110, human liver cell organoids111, or precision-cut liver slice cultures (PCLS)112 may yield insights into potential antifibrotic targets in human liver disease. PCLS are an ex vivo tissue culture system, which preserves the multicellular nature of the liver and maintains cells in their topographical niche, potentially generating more functionally relevant data112. PCLS have been successfully generated from rodent and human livers and are starting to be used to test the efficacy of antifibrotic agents113. In the future, these methods may be an invaluable addition to the assessment of potential antifibrotic therapies. The importance of precise patient stratification When a potentially effective antifibrotic therapy for chronic liver disease is identified, it is critical to select the appropriate trial candidates for treatment (Fig 3). The precise patient group will partly depend on the mode of action of the proposed treatment, for example inhibiting CCR9+ effector T cells is most likely to be effective for patients with PSC71 (see above), and therefore focussed, single aetiology studies are required. In contrast, treatments aimed at a “core” fibrosis pathway may be effective in multiple aetiologies of liver disease. However, as disease progression/regression varies widely between aetiologies, most studies focus on a single cause of liver disease. Furthermore, given the rapid rise in the prevalence of NAFLD, the majority of new antifibrotic clinical trials are being performed in this patient group (Table 2). Whilst this is appealing in terms of disease burden, the natural history of NAFLD poses significant challenges to trial design. Patients with NAFLD have multiple variables (such as age, ethnicity, gender, BMI, diabetes, genetic background, alcohol consumption, coffee consumption and other liver disease co-factors27) all of which may have profound effects on disease progression. Furthermore, NAFLD patients are often taking multiple other treatments (such as statins or ACE inhibitors) which could potentially influence liver fibrosis progression. Therefore, when designing new antifibrotic clinical trials, a great deal of care must be taken in terms of patient selection and the randomisation process, to try to ensure equal distribution of these multiple confounding factors. In order to elucidate whether an antifibrotic therapy is effective it could be argued that antifibrotic clinical trials should be undertaken in patients with established liver disease, who are at relatively high risk of developing liver-related complications. However, advanced liver cirrhosis will be inherently less reversible and more difficult to treat. In contrast, treating patients with earlier stage liver fibrosis may be more effective but, given the variable non-linear natural history of chronic liver disease, much more challenging to demonstrate clinical efficacy of potential therapies within a reasonable timeframe114. Furthermore, patients with earlier stage disease are likely to be asymptomatic and would almost certainly need to be treated with antifibrotic therapies for many years, in a manner analogous to blood pressure-lowering therapy for patients with hypertension. Hence, any new antifibrotic therapies will need to be extremely well tolerated to allow high levels of patient compliance.A further challenge to investigators is selecting an appropriate primary endpoint for their antifibrotic trial (Fig 3). To gain regulatory approval with the FDA, an antifibrotic therapy must improve how a patient “feels, functions or survives”. Given the lack of symptoms and protracted disease course in many patients with liver fibrosis, it is unlikely that these endpoints will be met within the timeframe of most clinical trials in this area. Hence, validated surrogate endpoints which predict long-term clinical outcomes are required. Currently, the “gold standard” surrogate endpoint is a paired pre- and post-treatment liver biopsy, demonstrating improvements in fibrosis/cirrhosis following the intervention. However, despite improvements in fibrosis quantitation on liver biopsy3, it reflects only a tiny proportion of the liver and is prone to significant sampling variability115. Thus, non-invasive biomarkers which accurately reflect the level of fibrosis in the whole liver and predict long-term outcome are essential. The current panel of widely available non-invasive liver fibrosis tests such as serum markers and transient elastography are useful in differentiating cirrhosis/advanced fibrosis from mild/no fibrosis, but are unlikely to be sensitive enough to detect more subtle changes in fibrosis stage following antifibrotic treatment. Newer methodologies which dynamically quantify fibrogenesis/fibrolysis may prove to be more useful. One such approach is the detection of ECM breakdown products in the serum, which is a more direct measure of ECM turnover, correlates with relevant clinical parameters such as hepatic venous pressure gradient (HVPG)116, and may be more dynamic, enabling monitoring of treatment effects. An alternative is to utilise imaging modalities such as radioimaging or MRI, alongside the administration of specific probes to ECM components such as collagen I117 or fibronectin118 , which may allow real-time assessment of fibrosis in the whole organ. Furthermore, if these new fibrosis imaging modalities are MRI-based, and therefore do not involve ionising radiation, whole organ quantitative fibrosis readouts could be taken longitudinally at multiple timepoints throughout a clinical trial, making liver biopsy, with its inherent limitations and risks, potentially redundant in this setting. Hopefully, in the coming years, cross-sectional imaging-based quantitative assessment of fibrosis will become a reality, both for diagnostic purposes and also as an extremely valuable tool in the area of antifibrotic trialling. ConclusionThe prevalence of chronic liver disease, with ensuant hepatic fibrosis and cirrhosis, is increasing worldwide. Liver transplantation is a highly effective treatment, however demand greatly outstrips donor organ supply, and this procedure is unavailable to the vast majority of patients on a global scale. Therefore, effective antifibrotic therapies are urgently required.The past three decades have seen huge advances in our understanding of the cellular and molecular mechanisms regulating liver fibrosis, although successful conversion of these scientific discoveries into tangible, potent anti-fibrotic therapies has proved significantly more challenging than perhaps was first thought. However, we now know that liver fibrosis is a highly dynamic and reversible process, displaying significantly more plasticity than virtually any other type of organ fibrosis, and so the development of antifibrotic and pro-regenerative therapies that harness and augment the inherent regenerative capacity of the liver should be a realistic and achievable aim. Encouragingly, the field continues to accrue an ever-lengthening list of attractive, potentially tractable therapeutic targets, however our current paradigm pipeline of in vitro assessment, followed by pre-clinical rodent model and then clinical trial has so far failed to deliver. A major reason for this is likely to be the significant disparity between pre-clinical models of fibrosis versus the highly complex biology of human fibrotic liver disease. In the coming years, we must re-align our ‘translational compass’ significantly more towards direct study of human fibrotic liver disease, encompassing all the various aetiologies and stages of human chronic liver disease. The new wave of ‘omics’ technology and ‘big data’ acquisition means we can now probe and understand the complexity of human fibrotic liver disease with ever-increasing depth and precision, pulling apart this process at a cellular and molecular level, allowing the identification of relevant therapeutic targets. Hopefully, by coupling these powerful new technologies with fastidious clinical trial design, and novel non-invasive modalities to longitudinally quantify fibrosis during clinical trials, we will see the successful bridging of the ‘translational gap’, and the introduction of effective antifibrotic therapies for patients with liver disease. Search strategy and selection criteria References for this review were identified through searches of PubMed with the search terms “liver fibrosis”, “hepatic fibrosis”, and “antifibrotic” from 1990 until July 2016. Articles were also identified through searches of the authors’ own files. Clinical trials were identified by searching using the search terms “liver” or “hepatic”, limited to interventional studies and with “fibrosis” in the outcome measures. Only papers published in English were reviewed. AcknowledgementsThe authors acknowledge the support of the Wellcome Trust and the Medical Research Council.Conflict of interestThe authors declare no conflict of interest.ContributorsBoth authors contributed equally to review design, literature searches, generation of figures and writing of the manuscript.References1.Nagula S, Jain D, Groszmann RJ, Garcia-Tsao G. Histological-hemodynamic correlation in cirrhosis-a histological classification of the severity of cirrhosis. 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J Hepatol. 2013; 59(5): 992-8.118.Chow AM, Tan M, Gao DS, Fan SJ, Cheung JS, Qiao Z, et al. Molecular MRI of liver fibrosis by a peptide-targeted contrast agent in an experimental mouse model. Invest Radiol. 2013; 48(1): 46-54.FIGURE LEGENDSFigure 1: The fate of activated myofibroblasts during liver fibrosis resolution. Summary of potential fates of activated myofibroblasts during hepatic fibrosis resolution and the key signals directing myofibroblast fate.Figure 2: Potential therapeutic targets for antifibrotic therapies in chronic liver disease. Antifibrotic therapies may inhibit fibrosis progression and/or promote fibrosis regression.Figure 3: Current challenges and potential solutions for effective translation of antifibrotic therapies in chronic liver disease. ................
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