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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 Author

Professor 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 4TJ

Tel: +0044 131 242 6653

E-mail: [email protected]

Summary

Chronic 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 reversible

Long 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 fibrosis

1. Epithelial cell damage

A 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.

2. Hepatic myofibroblasts

Myofibroblast 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 activation 26,

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 targets

Cytokines/Chemokines TGF-β

PDGF

CTGF/CCN2

Pro-inflammatory cytokines (e.g. TNF-α, IL-1β)

CCR2

CCR5

Membrane Receptors Cannabinoid Receptor (CB1)

Angiotensin II type 1 (AT1) receptor

Serotonin (5-HT2B receptor)

Endothelin-1

Nuclear Receptors Peroxisome-proliferator activator receptors (PPAR) –α, -δ and-γ

Vitamin D Receptor

Tyrosine Kinases Downstream signalling of range of growth factors

Adipokines Leptin

Adiponectin

Ghrelin

Pattern Recognition Receptors

TLR activation by intestinal dysbiosis

Interactions with ECM Increased matrix stiffness

ECM-induced integrin signalling

Autophagy Autophagy proteins

Reactive Oxygen Species NADPH Oxidases (NOX1, NOX2, NOX4)

Epigenetic changes DNA Methylation via DNA methyl transferases or MeCP2

Histone 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 apoptosis 28,

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) cells 30. 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.

3. Liver macrophages

In 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 tissue 48-50. 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.

4. Other immune cells

In 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.

5. 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 fibrosis

In 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 integrins

The 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 disease

As 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 clinicaltrials.gov 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 clinicaltrials.gov 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.

Treatment Biological target Aetiology of Liver Disease

Number of

Patients

Trial Outcome Reference or Clinicaltrials.gov

NumberReduce Epithelial Injury

GS-9450 Pan-caspase inhibitor HCV 307 Study terminated 00874796

Emricasan Pan-caspase inhibitor NAFLD 330 Ongoing 02686762

Post-transplant HCV

60 Results pending 02138253

Metformin Reduce insulin resistance NAFLD 80 Results pending 00134303

NAFLD 110 Reduced fibrosis 88

NAFLD 86 Results pending 02234440

Liraglutide GLP-1 agonist NAFLD 52 Reduced fibrosis progression

89

Exenatide GLP-1 agonist NAFLD 20 Study terminated 00529204


Oltipraz AMPK activator which reduces insulin resistance

NAFLD 83 No effect on fibrosis

90


MSDC-0602K Insulin sensitiser NAFLD 283 Results pending 02068339

Ethyl-eicosapentanoic acid

Anti-oxidant NAFLD 243 No effect on fibrosis

91

Metadoxine Anti-oxidant NAFLD 108 Ongoing 02541045

Omega-3 fish oils Anti-oxidant NAFLD 41 No effect on fibrosis

92


Obeticholic acid Farnesoid receptor X (FXR) agonist

NAFLD 283 Reduced fibrosis 93

NAFLD 2000 Ongoing 02548351

PBC 217 Results pending 01473524

PBC 350 Ongoing 02308111

PSC 75 Ongoing 02177136

Volixibat Apical Sodium-Dependent Bile Acid Transporter Inhibitor (ASBTi)


Aramchol Fatty acid-bile acid conjugate NAFLD 240 Ongoing 02279524

Metreleptin Leptin analogue NAFLD 20 Results pending 01679197

Inhibit Hepatic Myofibroblast Activation or Promote Myofibroblast Loss

Losartan

Irbesartan

Candesatan

Angiotensin II type 1 (AT1) receptor inhibitors


HCV 14 No effect on fibrosis

94

HCV 166 Results pending 00265642

ALD 85 Reduced fibrosis 95

Atorvastatin HMG-CoA reductase inhibitor NAFLD 150 Not reported 01987310

Pioglitazone

Rosiglitazone

Farglitazar

Peroxisome-proliferator activator receptor (PPAR) –γ agonist


96

HCV/HIV 31 Not reported 00742326


97

NAFLD 74 Reduced fibrosis progression

98


99


100


Elafibrinor Peroxisome-proliferator activator receptors (PPAR) –α and –δ agonist

NAFLD 276 Reduced fibrosis in elafibrinor responders

101


Benzafibrate PPAR –α, –γ and –δ agonist PBC 100 Results pending 01654731

Vitamin D Vitamin D Receptor NAFLD 200 Results pending 01623024


HBV 1500 Ongoing 02779465

Anti-CTGF monoclonal antibody

CTGF/CCN2 blockade HBV 228 Results pending 01217632

GS-4997 +/- Simtuzumab

ASK1 inhibitor +/- Anti-LOXL2 mAb


PRI-724 Wnt signalling inhibitor HCV 18 Pending 02195440

ND-L02-s0201 Vitamin A-coupled liposome targeting HSP47

Moderate to extensive fibrosis

25 Pending 02227459

Rimonabant CB1 receptor agonist NAFLD 165 Study terminated 00576667

NAFLD 89 Study terminated 00577148

Modulate Immune Responses

Pentoxyfylline TNF-α (pro-inflammatory cytokine) inhibition

NAFLD 55 Possible reduction in fibrosis

102

HCV 100 Not reported 00119119

IL-10 Anti-inflammatory cytokine HCV 30 Reduced fibrosis 103

Interferon-γ Immunomodulation HCV 502 No effect on fibrosis

104


105

HBV 99 Reduced fibrosis 106

PF-4136309 CCR2 inhibitor HCV 24 Study terminated 01226797

Maraviroc CCR5 inhibition HIV + coinfection with HBV and/or HCV

138 Results pending 01327547

Cenicrivroc CCR2/CCR5 inhibition NAFLD 289 Results pending 02217475

Viusid Nutritional supplement (anti-inflammatory and anti-oxidant)

HCV 100 Reduced fibrosis 107

Target Core Fibrosis Pathways

Pirfenidone TGF-β inhibition (along with anti-inflammatory effects and inhibition of HSP47)

HCV 150 Results pending 02161952

Hydronidone Pirfenidone derivative HBV 240 Ongoing 02499562

Simtuzumab Anti-LOXL2 mAb HCV +/-HIV 18 No effect on fibrosis

108

PSC 235 Results pending 01672853

Abbreviations: 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 research

Despite 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 biopsy 3, it reflects only

a tiny proportion of the liver and is prone to significant sampling variability 115. 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.

Conclusion

The 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

clinicaltrails.gov 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.

Acknowledgements

The authors acknowledge the support of the Wellcome Trust and the Medical Research Council.

Conflict of interest

The authors declare no conflict of interest.

Contributors

Both authors contributed equally to review design, literature searches, generation of figures and writing of the manuscript.

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FIGURE LEGENDS

Figure 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.

summary - university of web viewliver transplantation is an effective treatment for end-stage liver...

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