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Antifibrotic Therapies: Where Are We Now? Scott L. Friedman, MD1

Youngmin A. Lee, MD1

1 Division of Liver Diseases, Icahn School of Medicine at Mount Sinai,

New York, New York Semin Liver Dis 2016;36:87–98.

Abstract

Keywords

► liver fibrosis ► antifibrotic drugs ► nonalcoholic fatty liver disease ► nonalcoholic steatohepatitis ► hepatic stellate cell ► extracellular matrix

Address for correspondence Scott L. Friedman, MD, Division of Liver Disease, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, Room 11-70C, Box 1123, New York, NY 10029-6574 (e-mail: [email protected]).

Fibrosis is the wound-healing response of tissues to injury. Extensive characterization of organ fibrosis mechanisms has identified common core pathways in renal, pulmonary, skin, and liver fibrosis that offer novel antifibrotic approaches across tissues, in addition to organ-specific and/or disease-specific pathways. A growing number of small molecules and biologics have been identified that are reaching clinical trials for one or more fibrotic diseases, making new antifibrotic options for liver fibrosis an emerging reality. The accelerating pace of drug development, which will also include drug repurposing or combination therapies, heightens the need for novel methods for noninvasive fibrosis assessment without liver biopsy, which is critical to establishing surrogate endpoints for patients in clinical trials who have a low risk of hepatic decompensation. In this article the authors review mechanisms of liver fibrosis and outline potential therapeutic targets and antifibrotic therapies in preclinical studies and clinical trials.

Liver fibrosis is a dynamic process characterized by the accumulation of extracellular matrix (ECM) resulting from the wound-healing response to liver injury of any etiology. Chronic viral infection and liver injury due to nonalcoholic steatohepatitis (NASH), a more advanced subset of patients with nonalcoholic fatty liver disease (NAFLD), are the most common etiologies of fibrosis, and represent significant health burdens. Globally, both the incidence and prevalence of NAFLD are rising, with NAFLD now being recognized as the most common cause of chronic liver disease in the United States.1 Other etiologies include alcoholic liver disease; druginduced liver disease; and autoimmune, metabolic, and biliary disorders.2 Liver fibrosis reflects an imbalance that favors fibrosis progression (fibrogenesis) over regression (fibrolysis).3,4 Although fibrosis typically reverses after elimination of the causative injury, chronic persistent damage can result in highly cross-linked ECM and loss of potential reversibility.5 Cirrhosis is an advanced stage of fibrosis in which there is distortion of hepatic parenchyma and vascular structures.6,7 Cirrhosis can lead to decompensated liver disease, which is a major cause of morbidity and mortality worldwide. Manifestations of decompensated cirrhosis include portal hypertension with ascites, variceal hemorrhage, and/or hepatic encephalopathy. Additionally, cirrhosis is a “premalignant

Issue Theme New Treatments in Liver Disease; Guest Editors, Gregory J. Gores, MD, and Ariel E. Feldstein, MD

state” with a rising risk of hepatocellular carcinoma (HCC) over time; even patients with advanced fibrosis who are not yet cirrhotic have an elevated risk of HCC.8 Fibrosis had been regarded as an irreversible process, yet its potential reversibility was first suggested in 1979.9 More recently, there is increasing evidence for fibrosis regression now that therapies to cure or suppress ongoing liver injury have been developed. Even cirrhosis can reverse, especially as a consequence of successful antiviral therapy.10,11 Our understanding of the mechanisms underlying fibrosis has greatly advanced in the past 10 to 15 years, and a growing list of agents are being tested for their antifibrotic activity in animals and even humans; however, none of these has been approved yet for clinical usage. Although key pathways of hepatic stellate cell (HSC) activation and fibrosis are common to all etiologies of chronic injury, disease-specific pathways and patterns of fibrosis progression have been described.12 Regardless of whether targets are disease-specific or are present in all forms of liver injury, antifibrotic therapies for nonviral diseases will need to be effective in the face of ongoing injury because unlike in patients with viral hepatitis, the primary etiology may not be curable. Here we review the emerging concepts of liver fibrosis, both preclinical findings and emerging antifibrotic trials.

Copyright © 2016 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI http://dx.doi.org/ 10.1055/s-0036-1571295. ISSN 0272-8087.

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Young Joon Yoon, MD1

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Mechanisms of Liver Fibrosis: Clarification of Therapeutic Targets Liver Injury Hepatocellular injury associated with the presence of inflammation is a core element that initiates fibrosis. Recurrent or persistent damage to epithelial cells can then lead to progressive fibrosis.13 Epithelial cell death by either necrosis or apoptosis are both fibrosis drivers. Necrosis refers to the consequences of cell death characterized by rapid swelling and formation of membrane blebs, ultimately leading to cellular rupture. Both alcoholic liver injury and experimental injury due to carbon tetrachloride (CCl4) or acetaminophen are typically associated with significant necrosis and fibrosis.14,15 Apoptosis, a highly regulated type of cell death, is morphologically distinct from necrosis and is considered to be less inflammatory. Nonetheless, apoptosis of parenchymal cells can activate HSCs to initiate the fibrogenic cascade.16 Apoptotic stimuli include the pathway by the death receptors, Fas, and tumor necrosis factor-related apoptosis-inducing ligand.17,18 Knockout of Bcl-xL or Mcl-1, antiapoptotic mediators, promote hepatocyte apoptosis and can lead to fibrogenesis in experimental models.19,20 Fas signaling also enhances fibrogenesis through this pathway.21 In support of apoptosis’s role in fibrosis, inhibiting caspases attenuates fibrosis.22 Additionally, phagocytosis of apoptotic bodies by HSCs reduces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and is associated with fibrogenesis.23 The pan-caspase inhibitor Emricasan (IDN-6556) is effective in a murine model of NASH fibrosis24 and is being tested in patients with posttransplant hepatitis C virus (HCV) with results pending (ClinicalTrials.gov ID #NCT02138253).

Hepatic Stellate Cell Activation HSCs are resident nonparenchymal cells that normally lie in the subendothelial space of Disse. HSCs store retinyl esters in intracytoplasmic lipid droplets and maintain a quiescent, nonproliferative phenotype under normal homeostatic conditions. In normal liver, HSCs regulate retinoid metabolism, ECM homeostasis, and secrete mediators that may help preserve hepatocyte mass.25 In response to liver injury, HSCs become activated into proliferative myofibroblasts (MFBs) under the stimulation of many cytokines and soluble mediators. Activation of quiescent HSCs to proliferative MFBs is conceptually divided into two phases: initiation and perpetuation. Initiation is associated with rapid gene induction of key mitogenic and fibrogenic receptors resulting from paracrine stimulation and early changes in ECM composition, leading to a contractile and fibrogenic phenotype. Liver sinusoidal endothelial cells (LSECs) contribute to activation of HSCs by secreting tumor growth factor (TGF) β1 and platelet-derived growth factor (PDGF-) BB4 and can activate latent TGFβ into its active, fibrogenic form. Additionally, LSECs secrete fibronectin and other mediators that contribute to cellular crosstalk.26,27 HSC migration in response to mediators from LSECs is a key response to injury. In turn, HSCs can also activate LSECs by secreting angiopoietin-1 and vascular endothelial growth factor.28 Platelets are often overlooked as Seminars in Liver Disease

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a rich source of PDGF, TGFβ, and endothelial growth factor (EGF) during injury, yet they may be vital to the liver’s injury response. Immune cells, especially monocyte/macrophage subsets, have emerged as important determinants of fibrosis progression and regression. Subsets of monocytes may behave and migrate differently based on their distinctive chemokine receptor (CCR) phenotype. Monocytes derived from bone marrow quickly respond to inflammatory signals, migrate to inflamed tissue, and differentiate into macrophages. There are at least two subsets of monocytes in humans: CD14þþCD16 and CD14þCD16þ cells. CD14þþCD16 monocytes express CCR2, CD62L, and CD64.29 Corresponding monocytes in mice have been described: Murine Ly6Chi monocytes are similar to human CD14þþCD16 monocytes, and Ly6Clo cells are similar to CD16þ monocytes.30 CCR1 and CCR2 are more highly elevated in mouse Ly6Chi, whereas Ly6Clo monocytes highly express CCR5 and CX3CR1.31,32 Ly6Chi monocyte infiltration to inflamed tissue is dependent on CCR2 and CCR6.33 CCR2 and CCR6 are involved in monocyte migration from blood to inflamed tissue.34 CCL2, also known as monocyte chemoattractant protein 1 (MCP-1), is a ligand of CCR2. CCL2 drives CCR2þ Ly6Chi monocytes to injured liver, where monocytes are differentiated into profibrotic hepatic macrophages. CCL2 also promotes the release of immune cells from bone marrow during chronic liver injury.35 CCR1, CCR2, and CCR5 promote fibrosis in mice33,36 by mediating the migration of HSCs during liver injury and promoting the recruitment of macrophages and monocytes.37 A small molecule with dual inhibitory function against both CCR2 and CCR5, Cenicriviroc, was shown to be safe in a phase IIb trial of patients infected with HIV-1 (ClinicalTrials.gov ID #NCT01338883), and is being tested in phase II in patients with NASH fibrosis (ClinicalTrials.gov ID #NCT02217475). Cenicriviroc, which is orally available, may effectively block inflammatory signaling in HSCs, monocytes, and macrophages, thus inhibiting fibrogenesis, but clinical data are awaited. Hepatic macrophages produce cytokines including tumor necrosis factor α (TNFα), PDGF, interleukin (IL) 1β, IL6, and TGFβ. These cytokines further promote hepatocyte apoptosis and activate HSCs. Profibrogenic Ly6Chi macrophages can switch to proresolution Ly6Clo macrophages. Ly6Clo macrophages secrete fibrolytic matrix metalloproteinases (MMPs), including MMP-9 and MMP-13.38 CX3CR1 and its ligand fractalkine (CX3CL1) are among the key regulators of monocyte migration, differentiation and survival. For example, CX3CR1/ mice develop greater liver fibrosis than wild type (WT) mice following CCl4 treatment.39 Intrahepatic monocytes undergo preferential differentiation into proinflammatory macrophages in the absence of CX3CR1.40 Other inflammatory and immune cells, including T cells, natural killer cells, and dendritic cells, also affect HSC responses.40–42 Following their initial activation, progressive changes in HSCs lead to proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, and proinflammatory signaling. Initially thought to be preserved among all forms of liver

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yses on the effects of statins in preclinical (i.e., animal) models and clinical trials are needed to clarify their potential use as antifibrotics.67 Recent evidence has uncovered IL-22 and its antagonistic form IL-22 binding protein as another potential antifibrotic target in patients with chronic HCV.68,69

Biologic Activity of Extracellular Matrix In normal liver, the ECM is distributed between portal tracts, central veins, Glisson’s capsule, and the subendothelial space of Disse. Laminins, type IV collagen, and a mixture of proteoglycans are abundant ECMs in normal liver. In fibrotic tissues, fibril-forming type I and III collagens are abundant. Other matrix proteins such as fibronectin, osteopontin, hyaluronan, and proteoglycans are also increased in fibrosis. The ECM serves as a reservoir for cytokines and growth factors, and their release in response to injury promotes interaction between the ECM and neighboring cells. Among ECM-derived cytokines, VEGF promotes angiogenesis, hepatocyte growth factor (HGF) promotes epithelial cell proliferation and survival,70 and PDGF stimulates HSC proliferation.71 Other important growth factors that may be sequestered in the ECM include EGF and insulin-like growth factor. Membrane receptors mediate most ECM-HSC interactions. Prominent among these are integrins, which are transmembrane receptors composed of α and β subunits. They receive signals from ECM components to regulate cell adhesion, differentiation, proliferation, migration, and apoptosis. Blockage of αv-containing integrins by a small molecule (CVHM 12) in an experimental model led to a significant decrease of liver fibrosis in addition to renal and pulmonary fibrosis. Because this target contributes to fibrosis in several tissues, it may be considered a “core” fibrogenic pathway.72 Discoidin domain receptor (DDR) tyrosine kinase receptors that bind collagen ligands may also regulate cell–ECM interactions and cellular migration.73 Type I, type III, and type IV collagen promote HSC activation, proliferation, and migration. Relevant receptors for type I collagen are α1β1, α2β2, DDR1, and DDR2. DDR1 and DDR2 also mediate signaling between type III collagen and HSCs. Type IV collagen conveys signals through α1β1 and DDR1 receptors to activate, proliferate, and migrate. ECM accumulation leads to matrix stiffness, which also drives the activation of HSCs.74

Major Profibrotic Signaling Pathways TGFβ and its intracellular mediators, SMAD proteins, are the key fibrogenic signals in HSCs and portal MFBs.75 TGFβ1 binding and phosphorylation of the type I receptor induces phosphorylation of downstream SMAD proteins. The activated SMAD complex initiates the transcription of TGFβ1 target genes. TGFβ predominantly activates Smad3 in activated HSCs. In contrast, Smad7 acts in an autoregulatory negative feedback loop. TGFβ can also activate mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK),76 with significant cross-talk contributing to finetuning of intracellular signals.77 Seminars in Liver Disease

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injury, disease-specific pathways of HSC activation are increasingly recognized, especially in NAFLD.43 Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. Autocrine signaling by PDGF during HSC activation contributes to the rapid proliferation of HSCs.44,45 Vascular endothelial growth factor (VEGF) also promotes proliferation of HSCs and drives angiogenesis.46–48 Thrombin, EGF, keratinocyte growth factor, and basic fibroblast growth factor (FGF) are also identified as stellate cell mitogens.49,50 Chemotaxis, or directed migration of activated HSCs, leads to accumulation of cells within areas of injury and the formation of fibrotic septae. Chemoattractants include PDGF, TGFβ1, EGF, and MCP-1.51,52 Production of ECM, especially type I collagen, by activated HSCs is the main determinant of fibrosis. Transcriptional and posttranscriptional regulation of type I collagen synthesis has been extensively investigated. TGFβ1, through autocrine and paracrine stimulation, promotes ECM production. Connective tissue growth factor (CTGF/CCN2) is also a potent fibrogenic cytokine. Whereas CTGF is expressed at a low level in normal liver, its expression is highly enhanced in fibrotic liver specifically by HSCs. CTGF/CCN2 contributes to ECM production as well as the proliferation, migration, adhesion, and survival of cells.53 Biologics (i.e., antibodies), like FG-3019, a human monoclonal antibody directed against CTGF, have been tested in pulmonary fibrosis and are currently undergoing clinical testing in patients with hepatitis B virus (ClinicalTrials.gov ID #NCT01217632; ►Table 1). Contraction of activated HSCs may increase portal resistance during liver injury. Early changes in vascular reactivity may be reversible; however, progressive disruption of hepatic architecture to increase resistance to blood flow, combined with increased flow, lead to portal hypertension. Ca2þ-sensitive pathways dominate this response and Rho kinase activation phosphorylates myosin light-chain (MLC), leading to cellular contraction. Adenosine has an interesting role in injury because it is profibrotic, yet it inhibits stellate cell contraction via Rho antagonism.59 The actions of endothelin 1 are transduced through the Rho signaling pathway and promote HSC contractility. Rac, a small guanosine triphosphate (GTP) binding protein in the Rho family also mediates contraction of HSCs.60 Statins (HMG-CoA reductase inhibitors that reduce cholesterol) have pleiotropic effects including anti-inflammatory, antiproliferative, antioxidant, and immunomodulatory functions,61 which promote an antifibrotic effect as well. One recently proposed mechanism by which statins might exert antifibrotic activity is through a decrease in RhoA and Rho-kinase activity as a result of decreased synthesis of isoprenoids (e.g., geranyl-geranyl pyrophosphate), which serves as a lipid membrane anchor for Rho GTPase family members. Treatment of cirrhotic rats (CCl4 or BDL) with atorvastatin or simvastatin, respectively, led to a significant reduction in fibrosis.62,63 Antifibrotic effects were also analyzed in the lung64 and heart.65 A recent reanalysis of the HALT-C (Hepatitis C Antiviral Long-Term Treatment against Cirrhosis) trial cohort also showed that the use of statins was associated with a reduced risk for fibrosis progression in patients with chronic hepatitis C.66 Further anal-

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Table 1 Selection of antifibrotic and other representative trials registered at ClinicalTrials.gov Drug

Mechanism

Subjects

Results

Year of start

Phase

NCT identifier (alternate name)

Control primary disease UDCA

Secondary bile acid

PBC

Delayed fibrosis progression

______

4

54

Farglitazar

PPARγ agonist

HCV

No effect

2005

2

NCT0024475155

Pioglitazone/ Vitamin E

PPARγ agonist/ antioxidant

NASH

Improved NAS No progression in fibrosis

2003

3

NCT00063622 (PIVENS)56

Pioglitazone/ Vitamin E

PPARγ agonist/ antioxidant

NASH

Pending

2009

4

NCT01002547

GFT505

Dual PPARα/δ agonist

NASH

Pending

2012

2

NCT01694849

Obeticholic acid

FXR agonist

NASH

Improved NAS Improved fibrosis

2010

2

NCT01265498 (FLINT)

Obeticholic acid

FXR agonist

PBC

Pending

2014

3

NCT02308111

Obeticholic acid

FXR agonist

NASH

Pending

2015

3

NCT02548351 (REGENERATE)

Liraglutide

GLP-1 agonist

NASH

Pending

2010

2

NCT01237119

57

Antagonize receptor ligand interactions and/or intracellular signaling Irbesartan

Angiotensin II type 1 receptor antagonist

HCV

Completed Results pending

2005

3

NCT00265642

Losartan

Angiotensin II type 1 receptor antagonist

NASH

Completed

2010

2

NCT01051219 (FELINE)

Pentoxifylline

TNFα inhibitor

NASH

Improved NAS No change in fibrosis

2007

2

NCT 0059016158

GR-MD-02

Galectin-3 inhibitor

NASH

Completed Results pending

2013

1

NCT01899859

GR-MD-02

Galectin-3 inhibitor

NASH

Pending

2015

2

NCT02421094

IDN-6556 (Emricasane)

Pan-caspase inhibitor

Posttransplant HCV

Pending

2014

2

NCT02138253

Cenicriviroc

CCR5 and CCR2 antagonist

NASH

Pending

2014

2

NCT02217475 (CENTAUR)

PRI 724

Wnt signaling inhibitor

HCV

Pending

2014

1

NCT02195440

Pirfenidone

TGFβ and HSP 47 inhibitor

HCV

Pending

2014

2

NCT02161952

Nintedanib

Tyrosine kinase inhibitor

Liver cirrhosis

Pending

2014

1

NCT02191865

GS4997/ Simtuzumab

Apoptosis signalregulation kinase (ASK 1) inhibitor/ Anti-LOXL2 monoclonal antibody

NASH

Pending

2015

2

NCT02466516

HBV

Pending

2010

2

NCT01217632

Inhibit fibrogenesis FG-3019

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Anti-CTGF monoclonal antibody

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Table 1 (Continued) Drug

Mechanism

Subjects

Results

Year of start

Phase

NCT identifier (alternate name)

ND-L02 second-201

Vitamin A-coupled liposome targeting HSP 47

Moderate to extensive fibrosis

Pending

2014

1/2

NCT02227459

Simtuzumab (GS-6624)

Anti-LOXL2 monoclonal antibody

HIV and/or HCV

Pending

2012

2

NCT01707472

Simtuzumab (GS-6624)

Anti-LOXL2 monoclonal antibody

PSC

Pending

2012

2

NCT01672853

Abbreviations: CCR, chemokine receptor; CENTAUR, Combined Effects of Non-statin Treatments on Apolipoprotein A-I Up-Regulation (CENTAUR): A Feasibility Study; CTGF, connective tissue growth factor; FELINE, Anti-Fibrotic Effects of Losartan in NASH Evaluation Study; FXR, farnesoid X receptor; FLINT, The Farnesoid X Receptor (FXR) Ligand Obeticholic Acid in NASH Treatment Trial; GLP-1, glucagon-like peptide-1; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HSP, heat shock protein; LOXL2, lysyl oxidase-like molecule 2; NAS, NAFLD activity score; NASH, nonalcoholic steatohepatitis; PBC, primary biliary cirrhosis; PIVENS, Pioglitazone vs Vitamin E vs Placebo for Treatment of Non-Diabetic Patients With Nonalcoholic Steatohepatitis; PPAR, peroxisome proliferator-activated receptor; PSC, primary sclerosing cholangitis; REGENERATE, Randomized Global Phase 3 Study to Evaluate the Impact on NASH With Fibrosis of Obeticholic Acid Treatment; TGF, transforming growth factor; TNF, tumor necrosis factor; UDCA, ursodeoxycholic acid.

Toll-like receptors (TLRs) are transmembrane glycoproteins that recognize pathogen-associated molecular patterns (PAMPs), which are specific structural components of bacteria, fungus, and viruses. Toll-like receptors also respond to endogenous ligands, DAMPs, which are released during tissue injury. Ten TLRs (TLR 1–10) have been identified and TLR4 has been extensively investigated in liver; its exogenous ligand, lipopolysaccharide (LPS), is a component of the outer membrane of Gram-negative bacteria, but there are also endogenous TLR4 ligands, in particular high-mobility group box 1 (HMGB1). Toll-like receptors and their signaling molecules remain at low levels in healthy liver, whereas their expression is increased after liver damage.78 Activated HSCs express TLR2, TLR3, TLR4, TLR7, and TLR9.79 TLR4 signaling stimulates nuclear factor-κB (NF-κB) and JNK pathways to upregulate expression of chemokines and adhesion molecules.80 Single nucleotide polymorphisms (SNPs) of TLR4 can modulate downstream inflammatory and fibrogenic signaling.81 Dysbiosis due to dietary fat intake with consecutive alteration of the microbiome are thought to contribute significantly to the release of PAMPs and activation of TLRs in the pathogenesis of NAFLD-induced fibrosis, inflammation, and ultimately hepatocarcinogenesis.82 Recently, emulsifiers, commonly found in processed food, have also been implicated in promoting metabolic syndrome and chronic inflammatory diseases.83 Nuclear factor-κB is an important transcriptional regulator of inflammatory signaling and cell death during liver injury, fibrosis, and HCC development. Nuclear factor-κB plays a role in not only profibrogenic, but also in antifibrogenic signaling.84 LPS-mediated TLR4 activation transduces signal through NF-κB activation, leading to fibrogenesis. In contrast, NF-κB inhibits transcription of the α1(I) collagen gene.85 c-Jun N-terminal kinase contributes to HSC activation and fibrogenesis. Pan-JNK inhibitors decrease TGFβ and PDGF

signaling in human HSCs.86 In particular, JNK1-deficient mice have decreased fibrosis after BDL or CCl4.86 TGFβ and PDGF activate SMAD2 and SMAD3 via JNK, leading to HSC migration.87 NADPH oxidase (NOX) is a transmembrane enzyme complex that generates reactive oxygen species (ROS) in response to a range of stimuli. NADPH oxidase and NOX regulatory components are expressed on quiescent HSCs and are increased after cellular activation. In particular, activated human HSCs express increased catalytic subunits NOX1 and NOX2 and cytosolic regulatory factor p47phox.88 NOX-2-deficient mice develop less hepatic fibrosis compared to WT animals after BDL or CCl4.89 GKT-137831, an oral NOX4/ NOX1 inhibitor, attenuates liver fibrosis and hepatic apoptosis in these experimental models.90 GKT-137831, which has also been described as renoprotective, has been tested in a murine model of pulmonary fibrosis and is currently being assessed in a phase II trial for diabetic nephropathy (ClinicalTrials.gov ID #NCT02010242).

Epigenetic Regulation of HSC Activation Epigenetics refer to changes in gene expression and cell phenotype that are not caused by changes in DNA sequence; they can result from external or environmental factors. Epigenetic modifications include DNA methylation, histone modification, chromatin remodeling, and noncoding RNA. DNA methylation is common throughout the genome, but methylation of cytosine-phosphoguanine (CpG) dinucleotides in promoter regions attenuates gene expression.91 CpG methylation is regulated by DNA methyl transferases (DNMTs). DNMT-mediated phosphate and tension homolog hypermethylation activate HSCs.92 Histone acetylation is a reversible process associated with transcriptional activation, whereas histone deacetylation Seminars in Liver Disease

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results in transcriptional suppression. Several histone deacetylases (HDACs) regulate the activation of HSCs and promote liver fibrosis.93 Knock-down of HDAC4, HDAC5, and HDAC6 leads to induction of microRNA (miR-) 29, an antifibrotic microRNA, thereby inhibiting HSC activation.94 MicroRNAs are small noncoding RNA (22 nucleotides), which regulate posttranscriptional gene regulation. Profibrotic (e.g., miR-21) and antifibrotic miRs (e.g., miR-29, miR-16) are significantly regulated in HSCs (for a review, see95). Cell-specific delivery of inhibitory (e.g., anti-miR-21) or mimetic miRs (miR-29) are currently being evaluated in preclinical models of cardiac fibrosis and might offer a novel therapeutic approach. Mimics of miR-29 reverse experimental pulmonary fibrosis.96 Long noncoding RNAs are implicated in gene silencing, especially by DNA methylation. Maternally expressed gene 3 is downregulated in CCl4-induced mouse liver fibrosis models and human fibrotic liver.97

Resolution of Fibrosis If liver injury is acute and self-limited, accumulated ECM is eventually degraded. Reduction in the number of activated HSCs is critical to fibrosis regression. During resolution of fibrosis, activated HSCs may undergo apoptosis, senescence, or revert into an inactivated phenotype. Apoptosis of activated HSCs during resolution decreases HSC numbers.98 Both inhibition of NFκB and decreased TIMP-1 expression facilitate HSC apoptosis during the recovery phase following liver injury99,100 Senescence of HSCs via p53 activation contributes to reversion of fibrosis.101 Peroxisome proliferator-activated receptor γ (PPARγ), a member of the PPAR subfamily of nuclear receptors, is expressed in quiescent HSC; its expression is decreased upon HSC activation.102 Restoration of PPARγ promotes quiescence of HSCs in experimental systems.103 Metalloproteinases are enzymes that are critical to fibrosis regression by catalyzing physiological and pathological degradation of ECMs. After liver injury, activated HSCs produce MMPs, as well as their specific tissue inhibitors, the tissue inhibitors of metalloproteinases. In early liver injury, HSCs exhibit a matrix-degrading phenotype, but later their failure to degrade fibrillar collagen results in a net accumulation of ECM. HSCs and macrophages are key sources of MMP-2, MMP-9, and MMP-13. MMP-2 inhibits type I collagen production and promotes apoptosis of HSCs.69 Fragments of type XVIII collagen, peptide E4 (endostatin), not only prevent dermal and pulmonary fibrosis in mouse models, but also reduce existing fibrosis accompanied by reduced level of lysyl oxidase, an enzyme that cross-links collagen.104 Endostar, a human recombinant endostatin, with antitumorigenic and antiangiogenic properties, attenuates liver fibrosis in CCl4induced mice.105

Antifibrotic Therapies General Consideration: Preclinical Studies There are several considerations in developing molecules for antifibrotic therapy. Blocking core pathways of fibrosis that also contribute to tissue homeostasis outside the liver may Seminars in Liver Disease

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cause unwanted collateral effects in nonfibrotic extrahepatic tissues. Therefore, targeting molecules or pathways unique to the diseased tissue is optimal. On the other hand, targeting only one component of a complex signaling network may not be sufficiently potent, so that targeting more than one molecule is appealing.106 One consequence of attenuating HSC activation or cell number could be to deplete the liver of critical proregenerative mediators that have homeostatic activity in normal tissue, for example, by diminishing the expression of hepatocyte mitogens EGF or HGF. Although gene expression patterns between activated HSCs in culture and in vivo are similar, there are also notable differences. Therefore, targets interrogated in cultured cells must be validated in vivo. There are also differences in pathologic features and disease mechanisms between animal models and human disease, so that antifibrotic agents should be tested in more than one model to ensure that an effect is not restricted to only one model. Toxic agents such as CCl4 or thioacetamide are commonly used to induce liver injury and fibrosis in rodents. In these animal models, fibrosis first appears in the perivenular area, whereas in human liver, fibrosis is more commonly distributed in periportal and lobular areas. Genetically modified animal models, especially through targeted gene overexpression or silencing, have been widely used to investigate factors associated with liver fibrosis. In particular, HSC-specific gene silencing in mouse models can be achieved by using Cre recombinase expression under the control of a HSC-specific promoter, for example, the human glial fibrillary acid protein promoter.107 Lecithinretinol acetyltransferase (Lrat-) Cre transgenic mice also label most of the HSCs.108 Fate mapping in this model has demonstrated that HSCs are the dominant contributors of MFBs in models of toxic, cholestatic, and fatty liver disease. Drug delivery systems to target specific cells have been developed (for a review, see 13). For example, vitamin A-coupled liposomes targeting HSCs can deliver a potential antifibrotic drug into these cells while minimizing off-target effects.109,110

Moving toward Clinical Trials There is a growing concern about the value of animal models in predicting response to therapy in human liver diseases. One approach to overcome this limitation is to perform comprehensive transcriptomic analysis of animal models to determine whether they align with human disease gene expression datasets. There are a growing number of such gene array datasets in the public domain, and these resources are likely to expand considerably in the coming years, to include proteomics, epigenetics, and metabolomics data. Thus for example, this type of comparative analysis could not only identify the best animal models for specific human diseases or even for specific stages of liver disease, but could also establish the relevance of specific molecular targets uncovered in animal models to human disease. We are just at the beginning of a new era of big data analysis, and exploiting opportunities provided by these approaches is likely to greatly accelerate therapeutic target identification and validation of antifibrotic drugs.

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As noted, no animal models can fully represent the hepatic features of human liver disease. In addition to transcriptomic analyses, another means to overcome this gap is to start with human samples to reveal pathways associated with liver fibrosis.111 Immunodeficient mice with human hepatocyte transplantation have also been proposed to reproduce the pathological events in human liver disease, but these models are very costly and not amenable to higher throughput analysis.112 Culturing of human liver slices to model the liver ex vivo113 or “liver-on-a-chip” techniques might help close the gap.114 In addition to limitations of preclinical testing, translation of basic knowledge about liver fibrosis into clinical trials is confronting other challenges. Testing of antifibrotic drugs in noncirrhotic patients may need to be unrealistically lengthy if a reduction in clinical events is set as a trial endpoint, whereas drug trials in patients with advanced fibrosis may make it more difficult for a drug to yield a therapeutic response.115 The slow and nonlinear progression rate to advanced fibrosis requires the development of noninvasive biomarkers, because serial liver biopsies are not easily accepted by patients and providers. Moreover, biopsy is notoriously prone to sampling variability, and does not reflect the changes in the whole liver. At the least, quantitative assessment of collagen content by morphometry is preferable to discontinuous staging systems such as Metavir, Ishak, or NAFLD fibrosis scores, and they are much better correlated with the clinical outcomes.116 The development of noninvasive biomarkers as surrogates for liver biopsy that reflect changes in fibrogenic activity or fibrosis deposition is a high priority. Current noninvasive markers include assessment of stiffness by transient elastography, magnetic resonance elastography, or acoustic force radiation impulse imaging. Magnetic resonance imaging can also quantify inflammation and fibrosis, and assess collagen synthesis using nonradiolabeled isotopes.117,118 Positron emission tomography functional imaging may provide an estimation of functional cellular or metabolic activity, but not of the liver’s histology.38 Novel approaches in identifying biomarkers include stable isotope labeling of newly synthesized proteins with deuterated water (2H2O) in combination with mass spectroscopy to identify individual proteins and determine the half-life of collagen and other ECM proteins in the plasma of cirrhotic patients.119 A study of this type in HCV patients undergoing curative antiviral therapy is currently underway (ClinicalTrials.gov ID #NCT02438917). The diagnosis of cirrhosis may also be aided by analysis of the gut microbiome120 however, its sensitivity and specificity need to be established, and it remains to be determined if analysis of gut microbiota can also reflect the extent of fibrosis and/or fibrogenic activity. Serum tests, alone or in combination, have been intensively evaluated, especially in chronic hepatitis C, but their accuracy in NAFLD is not yet established.121,122 Serum markers can be classified either as indirect marker that represent liver inflammation and function, or direct markers that reflect ECM metabolism. These markers can be used as panels such as aspartate aminotransferase to Platelet Ratio

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Index (APRI), European Liver Fibrosis Test (ELF), Fib4, and FibroSure (FibroTest in non-U.S.). Although serum fibrosis markers may be useful to identify cirrhotics, they cannot clearly differentiate intermediate fibrosis stages.123 Moreover, serum markers may not reflect the amount of deposited connective tissue in liver.115 Subject selection and stratification are key elements in antifibrotic drug trial design. Risk factors such as genetic factors, age, gender, alcohol use, coffee consumption, and metabolic syndrome should be identified at the time of enrollment. Importantly, different therapies may have differing efficacy based on the stage of disease. For example, drugs targeting inflammation and cell injury are likely to have the most benefit at earlier and intermediate stages, whereas drugs targeting mechanisms that are more critical in advanced fibrosis may be better suited for testing in patients with cirrhosis.38 The slow, chronic course of liver disease requires drugs to be highly tolerable, and easily administered; thus, oral administration is preferred over parenteral therapies. However, in patients with more advanced disease at risk of clinical decompensation, parenteral administration of drugs might be acceptable.

Current Drug Trials Therapeutic approaches to fibrosis include several potential points of attack: (1) eliminate or control the underlying liver disease; (2) antagonize receptor-ligand interactions and intracellular signaling; (3) inhibit fibrogenesis; and (4) promote fibrosis resolution (see ►Fig. 1). Reduction of inflammation and modulation of the immune response are also important components of the therapeutic approach. A selection of ongoing clinical trials searched on ClinicalTrials.gov for “liver fibrosis” is listed in ►Table 1. Currently, more than 500 trials are registered and have been reviewed in detail recently.5

Control Underlying Liver Disease The farnesoid X receptor (FXR) ligand obeticholic acid improved all components of the NAFLD activity score as well as fibrosis in patients treated for up to 72 weeks (The Farnesoid X Receptor [FXR] Ligand Obeticholic Acid in NASH Treatment Trial; FLINT).57 Long-term benefits of this drug in patients with NASH fibrosis will be evaluated in a phase III trial (Randomized Global Phase 3 Study to Evaluate the Impact on NASH With Fibrosis of Obeticholic Acid Treatment; REGENERATE Trial, ClinicalTrials.gov ID #NCT02548351).

Antagonize Receptor-Ligand Interactions and Intracellular Signaling Antagonism of the 5-hydroxytryptamine (5HT) receptor in preclinical studies reduced fibrosis and enhanced hepatic regeneration.124 Metadoxine, a drug approved for alcoholic hepatitis, improved fibrosis in experimental murine models of liver fibrosis (CCl4 and BDL)125 and in HSCs.126 Preliminary data indicated Metadoxine might have beneficial effects in NASH127 and is currently being tested in a phase III trial for patients with NASH (ClinicalTrials.gov ID # NCT02541045). Protective effects are mediated by its antioxidant properties Seminars in Liver Disease

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Fig. 1 Therapeutic targets for antifibrotic agents. The framework for antifibrotic strategies include (1) Disease-specific therapies that control or cure the underlying disease. (2) Targeting receptor–ligand interactions to attenuate hepatic stellate cell (HSC) activation to mitigate fibrogenesis. (3) Inhibition of the most potent profibrogenic pathways, e.g., by preventing activation of latent TGFβ, or blocking the activity of CTGF, are currently in clinical evaluation. (4) Promote the resolution of fibrosis by enhancing the apoptosis of activated HSCs through the activity of either NK cells or with drugs and by increasing the degradation of extracellular matrix by preventing its cross-linking with antagonists to LOXL2 or by fibrolytic macrophages. FXR, farnesoid X receptor; PPAR, peroxisome proliferator-activated receptor; UDCA, ursodeoxycholic acid; SVR, sustained virological response; CB1, cannabinoid receptor type 1; ARB, angiotensin II receptor blocker; ET-1, endothelin 1; TGFβ, transforming growth factor β; CTGF, connective tissue growth factor; mAb, monoclonal antibody; NF-κB, nuclear factor kappa light chain enhancer of activated B cells; NK, natural killer; TIMP, tissue inhibitor of metalloproteinase; LOXL2, lysyl oxidase 2. (Reproduced from Lee et al. Pathobiology of liver fibrosis: a translational success story. Gut 2015;64(5):830–841 with permission)

and through restoration of NADPH, glutathione (GSH), and ATP levels, which may be partially mediated by 5HT-2B antagonism. A novel class of carbohydrate inhibitors against Galectin-3, GR-MD-02, showed significant antifibrotic activity in preclinical studies by reducing inflammation, fibrosis, and a number of Galectin-3-positive septal macrophages.128 Galectin-3 is important for HSC activation and fibrogenesis.129 A phase I clinical study testing GR-MD-02 in NASH patients was recently completed and phase II trials are underway.

Inhibit Fibrogenesis TGFβ is the best studied and most important fibrogenic cytokine, and its role in tissue fibrosis across many tissues is well established. However, because TGFβ also regulates homeostatic functions including growth suppression, systemic inhibition of TGFβ could enhance the development of Seminars in Liver Disease

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neoplasia. Therefore, selective blockade of the TGFβ pathway by targeting cell surface molecules involved in its activation are especially appealing. One example is an inhibitory antibody to αvβ6 integrin (an activator of latent-TGFβ1) (STX-100, ClinicalTrials.gov ID # NCT 01371305), which is being tested in idiopathic pulmonary fibrosis (IPF). Antifibrotic activity of another drug approved for IPF, pirfenidone, is also being tested in cirrhotics in a phase II clinical trial.130 Nintedanib, a tyrosine kinase inhibitor targeting VEGF receptor, FGF receptor, and PDGF receptor, is currently in a phase I clinical trial in patients with liver cirrhosis, having been recently approved in IPF as well.131

Promote Fibrosis Resolution Promotion of ECM degradation may be possible by targeting lysyl oxidase-like molecule 2 (LOXL2), since it promotes crosslinking of collagen and elastin.132 Clinical trials (phase

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Antifibrotic Therapies: Where Are We Now? II) using humanized monoclonal antibody targeting LOXL2 (Simtuzumab) in subjects with liver fibrosis (NASH) are underway (ClinicalTrials.gov ID #NCT 01672866). Very few clinical trials have been conducted with fibrosis as a primary endpoint, yet some encouraging findings have emerged. Pioglitazone and vitamin E demonstrated histologic benefit in some clinical trials, but weight gain associated with pioglitazone has restricted its appeal. Vitamin E has also only been tested in patients with NAFLD who do not have diabetes, so it cannot be widely recommended yet. Moreover, some reports suggest that high-dose vitamin E supplementation may have adverse effects.56,133,134

MCP miR MLC MMPs NADPH PAMPs PDGF PPAR TGF TLRs TNF VEGF WT

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monocyte chemoattractant protein microRNA myosin light chain matrix metalloproteinases nicotinamide adenine dinucleotide phosphate pathogen-associated molecular patterns platelet-derived growth factor peroxisome proliferator-activated receptor tumor growth factor toll-like receptors tumor necrosis factor vascular endothelial growth factor wild type

The detailed characterization of basic mechanisms in inflammation and fibrogenesis has unveiled potential novel therapeutic approaches that are being investigated preclinically. Clinical trials will progressively uncover effective compounds, but combination therapies should also be explored. Repurposing of drugs that are currently approved for other diseases may be attractive as another strategy for identifying antifibrotic compounds. In general, drugs targeting multiple pathways or regulating multiple components in core signaling are more appealing than compounds affecting only parts of a complex network. Also, because core fibrotic pathways of fibrosis are shared among other organs including the lungs and kidneys,135 drugs currently under investigation in other tissues might have potential in liver fibrosis as well. Due to the high prevalence of NAFLD, most clinical trials are currently focused on this disease; however, other chronic liver diseases such as autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or congenital hepatic fibrosis should not be overlooked, and disease-specific therapeutics for these conditions should be investigated as well.

Conflict of Interest Dr. Friedman is a consultant for the following companies: Abbvie Pharmaceuticals, Angion Biomedica, Blade Therapeutics, Blueprint medicines, Boehringer Ingelheim, Bristol Myers Squibb, Chemocentryx Therapeutics, Conatus, Debio Pharmaceuticals, DeuteRx, DS Biosciences, Eli Lilly Pharmaceuticals, Enanta Pharmaceuticals, Exalenz Biosciences, Fibrogen, Galectin Therapeutics, Galmed, Genfit, Immune Therapeutics, Intercept, Ironwood Pharmaceuticals, Isis Pharmaceuticals, Kinemed, Merck Pharmaceuticals, Nimbus Therapeutics, Nitto, Northern Biologics, Novartis, Ocera Therapeutics, Pfizer, Raptor Pharmaceuticals, Roche/Genentech, RuiYi, Sandhill Medical Devices, Scholar Rock, Shire Pharmaceuticals, Synageva BioPHarma, Takeda Pharmaceuticals, Teva Pharmaceuticals. Tobira Therapeutics, Viking Therapeutics, Zafgen.

Funding Work in the Friedman laboratory is supported by NIH Grants DK56621 and AA020709.

Abbreviations CCR CpG CTGF DDR EGF ECM ERK FGF GTP HCC HCV HGF HSC IL IPF JNK LSECs LPS MAPK

chemokine receptor cytosine-phosphoguanine connective tissue growth factor discoidin domain receptor endothelial growth factor extracellular matrix extracellular signal-regulated kinase fibroblast growth factor guanosine triphosphate hepatocellular carcinoma hepatitis C virus hepatocyte growth factor hepatic stellate cell interleukin idiopathic pulmonary fibrosis c-Jun N-terminal kinase liver sinusoidal endothelial cells lipopolysaccharide mitogen-activated protein kinases

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