Our understanding of the mechanisms and specific components underlying the development
and regression of liver fibrosis has matured toward clinical translation.[1 ] Specialized cell types such as activated hepatic stellate cells (HSCs) and myofibroblasts
(MFs)[2 ]
[3 ] are central effectors of fibrogenesis (see “Origin and Function of Myofibroblasts
in the Liver” by Wells and Schwabe in this issue), and other cells such as liver macrophages
can promote either fibrogenesis or fibrolysis in a context-dependent manner (see “Resolution
of Liver Fibrosis: Basic Mechanisms and Clinical Relevance” by Ramachandran, Iredale,
and Fallowfield in this issue). Moreover, the underlying etiology of chronic liver
damage determines both the mechanism and pattern of liver fibrosis, likely necessitating
different approaches to antifibrotic therapy (see below). Instead of mere quantification
of collagen and considering fibrosis as an endpoint, the dynamic processes of fibrogenesis
and fibrolysis—the de novo formation and removal of connective tissue, respectively,
that capture the dynamic nature of even advanced fibrosis— have taken center stage.
Tissue injury is the most common stimulus for fibrogenesis, and immediately results
in multiple coordinated processes aimed at initiating repair and regeneration, and
at activating host defense.[4 ] At early stages, initiating signals (DNA, adenosine triphosphate, other nucleotides
and adenosine), responding cells (macrophages, platelets, liver sinusoidal endothelial
cells [LSECs]), and soluble mediators (platelet-derived growth factor [PDGF], transforming
growth factor-beta [TGF-β]) induce concomitant wound-healing responses, initiating
repair, regeneration, and activation of host defense. With time, cells, cytokine responses,
and matrix components become more specialized, but continue to have potent interactions
with each other. Inflammation can either enhance the fibrogenic signal, for example,
via secretion of soluble mediators (interleukin [IL] 1-β, IL-13, IL-17, and PDGF-BB),
or induce fibrolysis (interferon- [IFN-] γ or IL-12). On the other hand, chronic inflammation
is often regulated and dominated by the immunosuppressive TGF-β1, which is a highly
potent fibrogenic factor. These interactions make inflammatory responses an attractive
target, and focused anti-inflammatory approaches are expected to reduce tissue injury
and fibrogenesis, without compromising liver regeneration, which is particularly attractive
in inflammatory pathologies such as alcoholic and nonalcoholic hepatitis.
The differences between individuals that determine why some repair with a scar-free
liver while others proceed to cirrhosis are determined by genetic and environmental
factors (“second hits”), and the quantity of these different contributing factors
appear to determine the outcome. Thus, the contribution of each cellular or signaling
pathway may vary between groups of individuals. However, from a therapeutic perspective
the situation seems manageable because the pathways that lead to fibrogenesis or induce
fibrolysis are common between individuals, and only differ quantitatively. It also
stresses the necessity of a personalized approach to treatment of fibrosis, using,
for example, several biomarkers that quantify key fibrogenic or fibrolytic pathways.
Notably, most of the pathways found for the liver are also central pathways in the
development or regression of fibrosis in other organs and vice versa.[1 ]
[5 ]
It is important to recognize that fibrolysis is as complex and dynamic a process as
fibrogenesis and provides additional therapeutic targets. Furthermore, cellular plasticity
with economy of cellular populations is a common organizing principle. This is best
demonstrated for liver macrophages that are key to the development of fibrogenesis
as well as fibrolysis (see review by Ramachandran et al in this issue). This makes
therapies that aim to delete cell populations deemed to be fibrogenic a blunt approach,
which is likely to also limit fibrolysis.
Recognition of the full spectrum of changes associated with severe liver fibrosis
is vital. In addition to quantitative and qualitative changes of the extracellular
matrix (ECM), including increased ECM crosslinking and stiffness, liver fibrosis is
associated with loss of hepatocytes, vascular remodeling, changes in cellular populations,
and overall architectural distortion. The regenerative capacity of the liver is a
great asset to all therapeutic strategies. However, therapies that aim to simply remove
the ECM may not be effective against all the other pathological changes, and could
even further impair liver function or increase the risk of liver cancer.
Preclinical Testing
In Vitro and In Vivo Models
In vitro models are necessary for early drug discovery to advance our understanding
of the molecular pathogenesis of liver fibrosis, and for high throughput testing once
a target has been identified.[7 ] These include culture-activated HSCs and HSC lines as well as other liver cells
that are contributory to the fibrogenic or fibrolytic process. However, advanced preclinical
proof of efficacy requires selected animal models, preferably mouse models that permit
assessment of antifibrotic efficacy in the complex multicellular context and provide
information on bioavailability, pharmacokinetics, pharmacodynamics, and toxicity.
Because these models are only an approximation to the human scenario, there has been
a tendency to omit a thorough in vivo preclinical validation before initiating larger
phase 2 clinical studies. Examples are the 2-year studies of interferon-γ and the
highly potent peroxisome proliferator activated receptor-γ (PPARγ) agonist Farglitazar
in patients with advanced-stage hepatitis C, which yielded no effect by state-of-the-art
biopsy-based fibrosis readouts.[8 ]
[9 ]
Animal models should reproduce the varied features of human liver fibrosis. These
features include the degree and pattern of inflammation, biliary versus parenchymal
damage, time course, and reversibility. Incorporation of the causative agent—hepatotropic
virus, alcohol, or metabolic syndrome—is ideal, but can only be achieved for some
disease or using humanized mice.[10 ] Although no single model will perfectly represent even a given human etiology, useful
predictions as to antifibrotic efficacy appear to be possible by using combinations.
Thus, mice that lack the hepatocyte phospholipid flippase Mdr2 provide a model of
spontaneous biliary fibrosis progression resembling primary sclerosing cholangitis,
and discontinuation of toxin-administration in advanced toxin-induced fibrosis mimics
advanced human parenchymal fibrosis with little tendency to reverse.[11 ]
[12 ] Both models are characterized by only low-level inflammation and therefore show
similarities to the target patients with advanced fibrosis of low-to-moderate inflammatory
activity. Drugs that work in both models (inhibiting progression and inducing regression,
respectively) may have a relatively high probability to be effective in man.
There has been significant progress in the development of rodent models of NASH. Earlier
models produced components of NASH including steatosis and inflammation.[13 ]
[14 ] Recently, diet-based models that use high-fat diets supplemented with cholesterol
and fructose have captured central features of NASH including the metabolic syndrome,
steatosis, inflammation, and fibrosis.[15 ]
An additional limitation is that the vast majority of studies are performed in a single
strain of mice (typically C57BL/6), yet there are significant differences in fibrosis
susceptibility between strains. Experiments are also typically done with young (6–12-week-old)
mice, whereas liver fibrosis is usually a disease of older age, with older age as
a risk factor for faster fibrosis progression.
Transgenic and Gene Deletion Models
Genetic models can confirm factors and mechanisms that drive fibrogenesis or fibrolysis
in vivo, for example, transgenic mice with overexpression of PDGF-B, PDGF-C, or TGFβ1.[16 ]
[17 ]
[18 ] However, these models do not reflect the multifaceted nature of human liver fibrosis,
and lack chronic inflammatory liver injury, a key component in the development of
fibrosis and long-term complications.[19 ]
Finally, in vivo models have to be done in an optimal and standardized quality, coupled
with fibrosis readouts that accord to state of the art. This includes (1) group sizes
of > 10 animals, (2) analysis of samples of sufficient size (5%–10% of the liver),
and (3) use of complementary quantitative fibrosis and fibrolysis readouts. Notably,
several past studies do not satisfy these criteria.[7 ]
Precision-Cut Tissue Slices
A criticism of animal studies is their unclear transferability to the humans, which
may vary with the pharmacological target. Human precision-cut tissue slices (PCTS)
that can be cultured for several days are ∼200-µm-thick punches of liver that partly
reflect the multicellular human context.[20 ]
[21 ] Precision-cut tissue slices can be obtained either from normal livers (resections,
spontaneous fibrogenic activation ex vivo) or from cirrhotic explants. Multiple drugs
can be tested in slices prepared from a small tissue block. This technology may serve
as a preclinical bridge between animal models and the patient setting. However, more
studies are needed for its validation.
One major obstacle is the species difference, with significant biological differences
between rodents and humans.[22 ] An approach to identify pathways that are important for fibrosis in humans is the
concept of core pathways that are required for fibrosis in multiple organs and species.[23 ] Increased testing of pathways in multiple organs in rodents is relatively straightforward,
and able to provide a greater degree of certainty that the pathway will be important
across different species. A second important issue is the high degree of homogeneity
in experimental models. The test and control populations in experimental models are
homogeneous across a wide range of parameters, including, age, sex, genetic background,
diet, microbiome, etc. None of these will apply to the eventual human population,
and it is relevant to ask if the efficacy of a compound as an antifibrotic is maintained
if there is a controlled break in homogeneity in experimental models.
The Immune Response as an Antifibrotic Target
The immune response interacts with fibrogenesis and fibrolysis at multiple points,
and is an attractive candidate for therapy.[24 ] The healthy liver is notable for a very vigorous innate and subdued adaptive immune
response.[25 ] Among the innate cell population, liver macrophages have been most thoroughly investigated
and have key functions in fibrogenesis and fibrolysis. The well-recognized resident
macrophage population of the healthy liver (Kupffer cells [KCs]) are present at birth
and are self-renewing.[26 ] After injury, KCs initiate a fibrotic response via recruitment of additional innate
immune cells, including large numbers of Ly6Chi inflammatory blood monocytes[27 ] that quickly acquire the macrophage phenotype CD11b+ F4/80+ ([Fig. 1 ]).[28 ]
[29 ]
[30 ]
[31 ] These infiltrating cells have the capacity to produce a wide range of cytokines,
many of which have potent proinflammatory or direct profibrotic actions on HSCs and
MFs, such as TNFα, IL-1β, TGF-β1, and PDGF-BB, respectively.[32 ]
[33 ] They also express a range of chemokines like CCL-2, CCL-3, CCL-5, CCL-7, and CCL-8,
which recruit MFs and other leukocytes.[34 ] Targeting some of these molecules promises to be an effective antifibrotic strategy.
To take TGF-β1 as an example, several strategies to block its activity have demonstrated
efficacy in rodent models of liver fibrosis. These strategies include a fully humanized
anti-TGF-β1 antibody (Lerdelimumab), soluble TGF-β1 receptors, blocking peptides,
and a small molecule to block downstream activin receptor-like kinase activity (SB431542;
NCT 00125385, 01665391, 01262001).[35 ]
[36 ]
[37 ]
[38 ]
[39 ] Similarly, inhibition of several chemokines and their receptors demonstrated antifibrotic
efficacy, including CCR5, CXCR4, and CXCR3 antagonists (NCT 00393120, 01413568).[40 ]
[41 ] A shared concern is that these mediators affect different cell types and are involved
in many processes including angiogenesis, and cellular proliferation and differentiation;
their inhibition may have significant off-target effects as well.[42 ] Some of these factors, especially chemokines, will also act differently if not in
an opposite, fibrolytic way upon removal of the primary insult. Liver macrophage populations
that have been vital for fibrogenesis undergo a major phenotypic switch, with enhanced
production of e.g., matrix metalloproteinases (MMPs) to degrade the excess ECM and
the release of proapoptotic ligands such as TRAIL, which can induce HSC and MF apoptosis.[43 ]
[44 ]
[45 ] These proresolution macrophages have a distinct phenotype (CD11bhi F4/80in tLY6Clow ) and gene expression profile.[32 ] For established fibrosis, enabling this phenotypic switch and enhancing the number
of proresolution macrophages is an attractive antifibrotic approach.
Fig. 1 Multiple interactions between immune and profibrogenic cells. The progression of
hepatic stellate cells (HSCs) from the quiescent to activated, to myofibroblasts,
and eventually apoptosis is greatly influenced by paracrine signals from infiltrating
blood monocytes which become tissue macrophages. At the initiation of injury, these
tissue macrophages provide activation and proliferation signals, and during the resolution
phase they provide apoptotic and reversion signals, but also actively digest and remove
excess extracellular matrix. Additionally, innate (natural killer) and adaptive (Th1,
Th2, and Th17) immune cells provide signals that can increase or decrease macrophage
mediated fibrogenesis. Indirect cytokine production is shown in brackets. IL, interleukin;
PDGF, platelet-derived growth factor; TGF, transforming growth factor.
The relative weight of the Th1 and Th2 T cell balance is an important determinant
of fibrosis for innate immune and T cells.[46 ]
[47 ] Thus the classically proinflammatory Th1 cytokines IFNγ and IL-12 are considered
antifibrotic/fibrolytic, whereas the Th2 cytokines IL-4 and IL-13 are profibrogenic.[47 ]
[48 ] The Th2 cytokines may be addressable by antibody-based therapies such as a bispecific
antibody targeting IL-4 and IL-13.[49 ] By analogy, macrophages can show a classical (M1) and an alternative (M2) polarization,
which is induced by the same or similar cytokines that also induce Th1 versus Th2
polarization.[50 ] However, there exist several subtypes of M2 macrophages, with some of them possibly
exhibiting antifibrotic effects, complicating simple Th1/M1 vs Th2/M2 polarizing approaches
using cytokine (blocking) approaches.[51 ]
[52 ] Therefore, skewing of this balance specifically toward Th1 (and M1) is more attractive
than general inhibition of the Th2/M2 pathway, although such an approach needs to
be balanced because it may enhance classical inflammation and tissue destruction.
The two related innate immune cell populations natural killer (NK) and natural killer
T (NKT) cells have opposite effects. Natural killer cells have an important role in
limiting fibrosis by inducing cell-cycle arrest and apoptosis of activated HSCs.[53 ]
[54 ] Conversely, depletion and adoptive transfer experiments suggest that NKT cells can
promote fibrogenesis, but the mechanism of their profibrotic action is not well characterized.[55 ] More recently, type 2 innate lymphoid cells (ILC-2), which resemble Th2 T cells,
have been demonstrated to be profibrogenic via secretion of IL13 and IL33, which directly
activate HSCs.[56 ]
All antifibrotic therapies, particularly those that exert a regulatory activity, need
to consider that the liver is never affected by fibrosis alone, but also by the underlying
(usually inflammatory) disease. In this respect, fibrosis needs to be addressed in
the context of the original disease. Antifibrotic therapies will affect many pathways.
To increase efficacy and reduce side effects, therapies for specific fibrotic diseases
will have to be well selected.
Regulating Platelet and Endothelial Function
Hepatic stellate cells are positioned adjacent to liver sinusoidal endothelial cells
(LSECs), and the two have close functional interactions.[57 ] After liver injury and the initiation of fibrosis are a loss of fenestrations in
LSECs, increased expression of vasoconstrictors (ET-1 and angiotensin II), and decreased
activity of vasodilators, most prominently nitric oxide (NO).[58 ] In addition to these classic vascular changes, LSECs contribute to deposition of
ECM (e.g., fibronectin and collagens I and IV), and cytokine production (e.g., TGF-β1
and PDGF-BB).[59 ] Liver sinusoidal endothelial cells can also respond to changes in sinusoidal shear
stress, with enhanced production of NO.[60 ]
[61 ]
[62 ]
Therapeutic targeting of LSECs in fibrosis has focused on their predominant role in
regulating the dynamic part of intrahepatic portal hypertension, which is a major
cause of morbidity and mortality in cirrhosis. Interventions have included broad spectrum
kinase inhibitors such as sorafenib or sunitinib, and inhibitors of vascular endothelial
growth factor and endothelial growth factor. Such interventions have resulted in changes
that go beyond the hemodynamic to include reduction of fibrotic matrix.[63 ]
[64 ] It is unclear how much of this reduction in fibrosis is due to regulation by LSECs,
and how much of it is due to non-LSEC actions of these agents. However, as in inflammation,
angiogenic mediators, while being profibrogenic during progression, can promote fibrolysis
during regression.[65 ] Liver sinusoidal endothelial cells also have a key role in regulating the relative
response between liver regeneration and fibrosis. This is due to a stromal factor
derived pathway, which can activate the chemokine receptors CXCR7 and CXCR4.[66 ] After acute injury, activation of the CXCR7 pathway with recruitment of the downstream
transcription factor Id1 results in a regenerative response. Chronic injury, however,
results in a persistent activation of the FGF receptor 1 in LSEC that dampens the
CXCR7-Id1 pathway, and activates a CXCR4 driven profibrotic pathway. Such pathways
that regulate the switch between regeneration and fibrosis are excellent candidates
for therapeutic intervention.[67 ]
Platelets are a rich source of profibrogenic factors, such as PDGF-BB and TGF-β1,
but the role of platelets in fibrogenesis had been understudied.[65 ]
[68 ]
[69 ] Recent reports have demonstrated that most if not all PDGF-BB in liver fibrosis
derives from activated platelets and that its specific inhibition with a therapeutic
antibody strongly attenuates fibrogenesis. Importantly, this effect is replicated
with aspirin,[8 ] a cheap and frequently used drug with an acceptable safety profile in early-to-moderate
stages of liver disease. This finding demonstrates that we can expect marked (synergistic)
antifibrotic effects by repurposing well-known drugs that are in use for other indications.
The ECM and Integrins as Antifibrotic Targets
A change in the composition and an increase in the amount of the ECM is the defining
feature of all forms of fibrosis. In the normal liver, the extracellular matrix is
composed predominantly of macromolecules including collagens (mainly the interstitial
types I, III, V, VI, and the basement membrane types IV, XV, XVIII, and XIX), and
a range of glycoproteins such as laminin isoforms and fibronectin, and several proteoglycans.[70 ]
[71 ]
[72 ] During the development of rodent and human cirrhosis, there is a 5- to10-fold increase
in the content of collagens, particularly of fibril-forming types I and III, and an
increase of elastin, laminins, and proteoglycans,[73 ] which is accompanied by more highly crosslinked collagen fibers. The total amount
of ECM is not only dependent on the rate of production, but also largely on the balance
between the matrix degrading MMPs, and the inhibitors of metalloproteinases (TIMPs),
especially TIMP-1.[31 ] The MMPs are a family of endopeptidases that are produced by a wide range of cells,
and taken together can degrade all the major constituents of the ECM.[74 ] The TIMPs reduce MMP functionality by several mechanisms including stabilizing the
proenzyme and also direct inhibition. Expression of TIMPs is more restricted than
that of MMPs, and is high in activated HSCs. Several experiments have shown that alteration
in either MMPs or TIMPs results in significant change in ECM deposition.
The ECM is not simply a downstream end product of the fibrotic cascade, but also directly
feeds back onto it.[71 ]
[75 ] An increase in the stiffness of the fibrotic matrix initially results in HSC and
MF activation via receptor- (mainly integrin) mediated signal transduction from the
altered ECM to the cellular cytoplasm and back to the ECM.[76 ] Integrin receptors that (1) sense the collagen matrix and collagen-derived fragments,
such as α1β1, α2β1, αvβ1, and αvβ3; (2) bind to fibronectin, such as αvβ3 and αvβ5;
or (3) release active TGF-β1 (αvβ6 and αvβ8), which plays an important role in fibrogenesis.[71 ]
[77 ]
[78 ] Taken together, these integrins and other ECM receptors mediate critical interactions
between the ECM and hepatic cell populations, resulting in functional changes including
adhesion, migration, proliferation, differentiation, and apoptosis, as well as modulation
of cytokine, chemokine, and growth factor mediated signaling.[71 ]
[79 ] Functional integrins are formed by noncovalent bonding of an α and a β subunit,
with 24 known members in humans.[80 ]
[81 ]
[82 ] In fibrosis, interest has focused on the role of αvβ6 and αvβ8 as activators of
extracellular stored latent TGFβ1, which is proteolytically processed to active TGF-β1,
for example, via MMP-14 mediated cleavage, upon cellular contraction and stretching.[71 ]
[83 ]
[84 ]
[85 ] Latent TGFβ1 is tethered to αvβ6 or αvβ8 on activated cholangiocytes or HSCs/MFs,
respectively via an arginine-glycine-aspartic acid motif.[77 ]
[86 ]
[87 ] Integrin αvβ6 is virtually absent in the healthy liver and highly expressed after
a range of insults.[86 ]
[88 ]
[89 ] Therefore, the relative cellular specificity of the αv and especially TGF-β1 activating
integrin αvβ6 permits selective inhibition of TGF-β activity in areas of mechanical
stiffness and associated fibrogenesis. This is vital as total inhibition is known
to result in unwanted proinflammatory changes.[90 ] More generally, the family of αv integrins is expressed on many liver cell populations;
genetic deletion or pharmacological inhibition of all αv integrins results in attenuated
fibrogenesis,[91 ] or in the abundant integrin αvβ3 (and αvβ5) that is mainly expressed on HSCs/MF
and macrophages.[92 ]
[93 ]
Collagens, the major ECM proteins in fibrosis, and elastin are stabilized via enzymatic
crosslinking, which confers resistance to degradation, and thus may limit reversibility
of established fibrosis.[94 ] There has been a focus on the family of lysyl oxidases (LOX) that crosslink fibrillary
collagen mainly at the nontriple helical ends (telopeptides) of the collagen molecules.[11 ]
[95 ] LOX enzymes constitute a family of five members: LOX and LOX-like (LOXL) 1–4. They
are secreted, copper-dependent amine oxidases with a variable N-terminal region and
a conserved C-terminal domain that is necessary for catalytic activity. Expression
of the LOX proteins is tightly controlled in a time- and organ-dependent manner during
development, but aberrant expression and activity of these enzymes has been reported
in a range of diseases associated with the ECM and in cancers,[96 ]
[97 ] including an upregulation of LOX and LOXL2 in Wilson's disease, primary biliary
and other etiologies of cirrhosis, and in pulmonary fibrosis.[98 ]
[99 ]
[100 ] Hepatic stellate cells and portal MFs are major producers of LOX and LOXL2 in the
liver.[101 ] A humanized antibody (Simtuzumab) that blocks LOXL2 activity is currently being
assessed in a large clinical study for liver fibrosis in patients with PSC or NASH
(NCT01672853, NCT01672866, NCT01672879).[99 ]
Targeting Fibrosis Reversal
Recent animal studies have revealed that during experimental fibrosis regression up
to half of the myofibroblasts undergo senescence and apoptosis, whereas the rest acquire
a quiescent phenotype.[102 ]
[103 ] The factors governing the inactivation of myofibroblasts are under investigation.
For example, PPARγ plays a (limited) role in the re-establishment of the quiescent
HSC phenotype,[102 ] while matrix stiffness[104 ] and crosslinking is currently addressed by LOXL2 inhibition (ClinicalTrials.gov,
NCT01452308).[99 ]
Recruitment and activation of monocytes/macrophages is central to both fibrogenesis
and fibrosis regression in rodents.[105 ] Although targeting macrophage recruitment or polarization would be an attractive
approach, the functional heterogeneity of macrophage subpopulations in humans has
not yet been adequately characterized. Thus no clear links can be made yet from animal
studies to human disease and the macrophage subsets may be dependent on the etiology
of the liver disease. One rational attempt is the use of chemokine antagonists whose
role in fibrogenesis seems to be preserved among species. Therefore, preventing the
early recruitment of profibrotic mononuclear cells by CCL2 inhibition intrahepatic
macrophages may be shifted toward the “restorative” subset, accelerating fibrosis
regression.[106 ]
So Many Targets: Which Ones Are Attractive for Further Clinical Development?
[Fig. 2 ] illustrates the complexity of cellular interactions and fibrogenic or fibrolytic
signals exchanged between these cells. For the past 20 years there has been a steady
addition to the number of molecules and pathways that are targets for antifibrotic
therapy. TGFβ1 is one of the earliest such molecules and still occupies center stage.
However, systemic inhibition of TGFβ1 results in increased inflammation.[107 ] This spurred the targeting of specific steps in TGFβ1 activation, in a localized
manner. Inhibition of integrin αvβ6, with reduction of TGFβ1 activation promises to
be a highly effective and localized antifibrotic approach,[86 ]
[88 ]
[89 ] and clinical trials using antibodies against avβ6 are underway.[86 ] Connective tissue growth factor (CTGF) amplifies TGFβ1 signaling, and a monoclonal
antibody targeting CTGF has shown promise in animal models of pulmonary fibrosis.[108 ]
Fig. 2 Multicellular context of fibrogenesis and fibrolysis: The postulated major cellular
functional units and secreted factors that should be addressed in their complexity
when designing effective antifibrotic strategies. (A ) Vascular and (B ) biliary unit. Profibrogenic targets are underlined, in contrast to putative fibrolysis-inducing
targets in italics and red. Profibrogenic targets are underlined, in contrast to putative
fibrolysis-inducing targets in italics. Modified from Schuppan and Kim.[1 ] Baso, basophil; CCL, CC chemokine ligand; CTGF, connective tissue growth factor;
CXCL, CXC chemokine ligand; ET-1, endothelin-1; HGF, hepatocyte growth factor; IFN,
interferon; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase;
NO, nitric oxide; PDGF-BB, platelet-derived growth factor with two subunits B (in
parenthesis because a recent study indicates that most if not all PDGF-BB in liver
fibrosis derives from activated platelets[12 ]; PMN, polymorphonuclear neutrophil; ROS, reactive oxygen species; TNFα, tumor necrosis
factor α; Shh, sonic hedgehog; TGFβ1, transforming growth factor β1; Th, T helper
cell; TIMP, tissue inhibitor of metalloproteinases; TRAIL, TNF-related apoptosis-inducing
ligand; Treg, regulatory T cell.
Attenuating the activated phenotype of myofibroblasts is an attractive approach due
to their key role in ECM deposition. Inhibition of the cannabinoid receptor 1 (CB1)
reverses myofibroblast activation and attenuates experimental liver fibrosis.[109 ] This has passed the proof of principle state, and peripheral-acting CB1 antagonists
that may circumvent adverse side effects on the central nervous system like depression
are being developed.[110 ] In fibrotic NASH, progression is intimately linked to insulin resistance/type 2
diabetes, and the associated lipotoxic hepatocyte death and intestinal dysbiosis,
providing rational targets for both antiinflammatory and antifibrotic therapy in this
condition.[111 ]
[112 ] Therapeutic strategies include reducing oxidative stress, improving insulin signaling,
activating the farnesoid X receptor receptor (e.g., with obeticholic acid), fibrosis-targeted
inhibitors of hedgehog signaling, combined peroxisome proliferator activated receptor
(PPAR)α/δ agonists,[113 ]
[114 ]
[115 ] or manipulation of the altered gut microbiota using probiotics or microbiota transfer.[112 ]
[116 ]
Oxidative stress is an important cofactor in fibrosis, but the use of antioxidants
has been disappointing.[117 ] This may be due to differences between animal models and human disease, and the
fibrosis stage and cell-specific regulation of oxidant and antioxidant pathways. Activation
of NADPH oxidases (NOX1, NOX 2, and NOX4) induces HSC activation[118 ]
[119 ]
[120 ] NOX4 can trigger apoptosis in hepatocytes.[120 ] Inhibition of NOX1/NOX4 suppresses fibrogenesis in the CCl4 and bile duct ligation models, in pulmonary[120 ]
[121 ]
[122 ] and in interstitial kidney fibrosis. A phase II trial is underway in diabetic kidney
disease (ClinicalTrials.gov NCT02010242).
[Tables 1 ] and [2 ] list relevant clinical drug trials using antifibrotic agents in liver fibrosis or
other organ fibrosis with fibrosis as the primary or coprimary endpoint. What is remarkable
is the diversity of agents that have been tested. They range from drugs with very
broad or poorly characterized mechanism (e.g., omega-3 fats and vitamin D), to specific
receptor inhibitors (losartan and liraglutide), broad but fairly low intensity anti-inflammatory
and antiapoptotic effects (pentoxifylline and ursodeoxycholic acid), or multikinase
inhibitors (nintedanib). This is a reflection of the wide range of biological processes
that are involved in the development of liver fibrosis. Due to the obvious concerns
of redundant pathways, and individual heterogeneity in active pathways that lead to
fibrosis, there is a significant risk that many of the single agents listed may not
have significant efficacy and/or display off-target side effects. However, the past
and current studies are already providing a rich resource for designing effective
treatments that would also exploit drug combinations in the near future. Notably,
two antifibrotics (pirfenidone and nintedanib) have recently been approved by the
Food and Drug Administration and the European Medicines Agency for the treatment of
pulmonary fibrosis.
Table 1
Major studies with liver fibrosis as primary or coprimary endpoint (studies with at
least 50 patients)
Cause
Drug name (action), Treatment, Patients included (F,C, NR,SVR)
Efficacy
Year of completion/publication
Phase
No. of patients
NCTRef
HCV (not exclusively antiviral agents)
Pentoxiphylline (anti-TNFα) vs. vit E; 1 y, r, db (F)
No results reported
2006
3
100
00119119
IFNα-2b + R vs. IFNα-2b + R + Viusid (ascorbic acid, zinc, glycyrrhizic acid); 48 wk,
r (F/NR)
Improved fibrosis score
2007
−
100
[124 ]
Farglitazar (PPARγ agonist); 52 wk, r, db (F/NR)
No effect
2008/2010
2
225/265
00244751[9 ]
GS-9450 (pan-caspase inhibitor) vs. plac; 24 wk, nr, db (F/NR)
No results reported
2010
2
307
00874796
Irbesartan (AT1R antagonist) vs. plac; 2 y, r, db (F/NR)
Pending
2013
3
166
00265642
Fuzheng Huayu (Chinese herbal drug) vs. plac, 48 wk, r, db (F)
Pending
2014
2
100
00854087
Pirfenidone (anti-inflammatory) vs. plac 2-year intervention
Pending
2014
2–3
150
02161952
HBV (not exclusively antiviral agents)
Salvianolic acid B (ingredient of Fuzheng Huayu) vs. IFNγ; 6 mo, r, db (F)
No effect
2002
−
60
[125 ]
Fuzheng Huayu) vs. plac; 6 mo, r, db (F); biopsy and serum fibrosis markers
Significant for fibrosis regression and fibrosis markers
2005
226
[126 ]
FG-3019 (anti-CTGF mAb) vs Entecavir vs. plac; 45 wk, r, db (F)
Pending
2016
2
228
01217632
Entecavir ± Fuzheng Huayu (Chinese herbal drug) vs. plac, 48 wk, r, db (C)
Pending
2016
4
700
02241590
HBV/HCV coinfected
Oltipraz (antiproliferative agent) vs. plac; 24 wk, r, db (F,C)
No effect
2007/2011
2
83
00956098
PBC
UDCA (hydrophilic bile acid) vs. plac; 2 y, db (F,C)
No effect
1991
3
146
[127 ]
UDCA vs. plac; 4 y, r, db (F,C)
Lower fibrosis progression rate;
2000
4
103
[128 ]
Obeticholic acid (FXR agonist) vs. plac; 12 mo–8 y, r, db (F); UE and serum fibrosis
markers
Pending
2023
3b
350
02308111
Alcoholic hepatitis
Candesartan (ACE inhibitor); 6 mo, r, db (F)
Histological improvement; 33.3% vs 11.6% (p = 0.020)
2009/2012
1/2–2
85
00990639[129 ]
PSC
GS-6624 (anti-LOXL2 mAb) vs. plac; 96 wk, r, db (F)
Pending
2015
2
225
01672853
NASH
Orlistat (pancreatic lipases inhibitor) vs. 1400 kcal diet (30% fat); 36 wk, r, ol
(F)
No results reported
2006
4
50
00160407
Pioglitazone (PPARγ agonist) vs. plac; 6 mo, r, db
No effect
2006
4
55
00227110[130 ]
Pioglitazone vs. plac; 1 y, r, db (F)
Decreased fibrosis progression
2008
−
74
[131 ]
Pioglitazone vs. vit E vs. plac; 2 y, r, db (F)
Trend for decreased fibrosis progression for Pio groups
2009/2010
3
247
00063622[132 ]
Rosiglitazone (PPARγ agonist) vs. plac; 1 and 2 y, r (F)
No effect on fibrosis
2010
−
53
[133 ]
Pentoxifylline (anti-TNFα) vs. plac; 1 y, r, db (F)
Improved steatosis, lobular inflammation and fibrosis
2010/2011
2
55
00590161[134 ]
Rosiglitazone (Rosi) vs. Rosi + Metformin vs. Rosi + Losartan; 48 wk, r, ol (F)
No effect on fibrosis
2011
−
137
[135 ]
High-dose UDCA vs. plac, 1 y, r, db (F)
Significant reduction only of FibroTest
2011
3
126
[136 ]
Metformin (AMP kinase activator, antidiabetic); 1 y, r, db (F)
No results reported
2012
4
80
00134303
Metformin vs. insulin; 1 y, r, (C)
Pending
2016
126
NCT02234440
Liraglutide (GLP-1 agonist) vs. plac; 48 wk, r, db (F)
No results reported
2013
2
52
01237119
Pentoxifylline + vit E vs. vit E; 3 mo (biopsy), r, db (F)
No results reported
2013
3
120
01384578
Losartan (AT1R antagonist) vs. plac; 2 y, r, db (F)̀
Pending
2014
3
214
01051219
Obeticholic acid (FXR agonist) vs. plac; 72 wk, r, db (F)
Significant for steatosis, lobular inflammation; marginally significant for fibrosis
2014
2
280
01265498[137 ]
Pioglitazone (PPARγ agonist) vs. vit E vs. plac; 1.5 and 3 y, r, db (F)
Pending
2014
4
90
00994682
GS-6624 (anti-LOXL2 mAb; 75 mg vs. 125 mg) vs. plac; 100 wk, r, db (F)
Pending
2015
2
225
01672866
GS-6624 (200 mg vs. 700 mg) vs. plac; 100 wk, r, db (F,C)
Pending
2015
2
225
01672879
GFT505 (dual PPAR α/δ agonist); 52 wk, r, db (F)
Pending
2015
2
270
01694849
Pioglitazone (Pio) vs. vit E vs. vit E + Pio vs. plac; 1.5 and 3 y, r, db (F)
Pending
2015
4
90
01002547
Vit D vs. lifestyle counseling; 2 y, r, ol (F)
Pending
2014
3
200
01623024
Vit D vs. plac; 48 wk, r, db (F)
Pending
2015
2
60
01571063
Omega-3 (fish oil) vs. plac; 1 y, r, db (F)
No results reported
2010
2/3
64
00681408
Omega-3 (fish oil); 18 mo, r, sb (F)
No results reported
2013
2
100
00760513
Docosahexaenoic acid; 2 y, r, db (F)
No results reported
2011
1/2
60
00885313
Eicosapentaenoic acid vs. plac; 1 y, r, db (F)
No results reported
2012
2
243
01154985
Diamel (dietary supplement) vs. plac vs. lifestyle counseling; 52 wk, r, db (F)
No results reported
2012
3
158
00820651
Polypill (atorvastatin, valsartan); no biopsy (UE); 5 y, r, ol (F)
No results reported
2018
3
1500
01245608
NASH Surgery
Bariatric surgery (meta-analysis of 21 cohort studies) (F,C)
Variable effect
2010
−
1643
[138 ]
Abbreviations: ACE, angiotensin-converting enzyme; AT1R, angiotensin II receptor type
1; C, cirrhosis; CTGF, connective tissue growth factor; db, double-blind; F, fibrosis;
FXR, farnesoid X receptor; GLP-1, glucagon-like peptide-1; IFN, interferon; IL, interleukin;
LOXL2, lysyl oxidase-like 2; mAb, monoclonal antibody; NCT, number at ClinicalTrials.gov;
nr, nonrandomized; NR, nonresponders; ol, open-label; plac, placebo; r, randomized;
retro, retrospective analysis; TNFα, tumor necrosis factor α; UDCA, ursodeoxycholic
acid; UE, ultrasound elastography; vit, vitamin.
Table 2
Studies in pulmonary and other fibrosis with fibrosis as primary or coprimary endpoint
(studies with at least 50 patients)
Fibrosis
Drug name/Treatment
Efficacy
Year of completion/ publication
Phase
No. of patients
NCTRef.
Pulmonary
Etanercept (anti-TNFα) vs. plac; 48 wk, r, db
No effect
2005/2008
2
88
00063869[139 ]
N-acetylcystein (NAC, antioxidant) vs. plac; 1 y, r, db
Worsening of FVC and DLCO in NAC-arm, no change in mortality
2005
1/2
182
[140 ]
Bosentan (dual ET-1AR and ET-1BR antagonist) vs. plac; 1 y, r, db
Bosentan vs. plac; 12, 21 and 3 y (biopsy), r, db
Worsening of PFT; decline in FVC, DLCO and O2 saturation.
No significant effect
2005/2008
2010/2011
2/3
3
158
616
00071461
00391443
Imatinib (kinase inhibitor) vs. plac; 92 wk, r, db
No effect
2010
2/3
120
00131274[141 ]
Ambrisentan (ET-1AR antagonist) vs. plac; 92 wk, r, db
Terminated due to lack of efficacy
2012
3
600
00768300[142 ]
Pirfenidone (anti-TGFβ, anti-TNFα, anti-IL-1β) vs. plac, 72 wk, r, db
Pirfenidone vs. plac; 52 wk, r, db
Pirfenidone vs. plac; 52 wk, r, db
Study 004: reduced decline in FVC with high-dose pirfenidone
2008
3
435
00287716[143 ]
Study 006: no difference in FVC
Significant worsening of FVC
Improved FVC, no difference in survival
2008
2010
2014
3
3
3
344
275
555
00287729[143 ]
[144 ]
[145 ]
BIBF1120 (Nintedanib, multi-RTK inhibitor) vs. plac; 1 y, r, db
BIBF1120 vs. plac; 52 wk, r, db
BIBF1120; 3 y, nr, ol
Significantly reduced FVC decline and incidence of exacerbations
Significantly reduced FVC decline
Pending
2011
2014
2015
2
2
432
1066
198
00514683[146 ]
01170065
CNTO888 (anti-MCP1/CCL2 mAb) vs. plac; 74 wk, r, db
No results reported
2012
2
126
00786201
QAX576 (anti-IL13 mAb); 4 wk, nr, ol
No results reported
2009
2
52
00532233
FG-3019 (anti-CTGF mAb); 109 wk, ol
Pending
2014
2
84
01262001
Myelofibrosis
GS-6624 (anti-LOXL2 mAb); 24 wk, r, ol
Pending
2014
2
54
01369498
Abbreviations: AT1R, angiotensin II receptor type 1; CTGF, connective tissue growth
factor; CXCR2, CXC chemokine receptor type 2; db, double-blind; DLCO , diffusing capacity of the lungs for carbon monoxide; ET-1A(B)R, endothelin-1 receptor
type A(B); FVC, forced vital capacity; HMGCoAR, 3-hydroxy-3-methyl-glutaryl-coenzyme
A reductase; LOXL2, lysyl oxidase-like 2; mAb, monoclonal antibody; MCP1/CCL2, monocyte
chemoattractant protein-1/CC chemokine ligand-2; mTOR, mammalian target of rapamycin;
NCT, number at ClinicalTrials.gov; nr, nonrandomized; ol, open-label; PFT, pulmonary
function test; plac, placebo; r, randomized; RTK, receptor tyrosine kinase; TNFα,
tumor necrosis factor α.
