Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies

Targeting Liver Fibrosis: Strategies for Developmentand Validation of Antifibrotic Therapies

Yury Popov and Detlef Schuppan

We have made striking progress in our understanding of the biochemistry and cell biologythat underlies liver fibrosis and cirrhosis, including the development of strategies and agentsto prevent and reverse fibrosis. However, translation of this knowledge into clinical practicehas been hampered by (1) the limitation of many in vitro and in vivo models to confirmmechanisms and to test antifibrotic agents, and (2) the lack of sensitive methodologies toquantify the degree of liver fibrosis and the dynamics of fibrosis progression or reversal inpatients. Furthermore, whereas cirrhosis and subsequent decompensation are accepted hardclinical endpoints, fibrosis and fibrosis progression alone are merely plausible surrogates forfuture clinical deterioration. In this review we focus on an optimized strategy for preclinicalantifibrotic drug development and highlight the current and future techniques that permitnoninvasive assessment and quantification of liver fibrosis and fibrogenesis. The availabilityof such noninvasive methodologies will serve as the pacemaker for the clinical developmentand validation of potent antifibrotic agents. (HEPATOLOGY 2009;50:1294-1306.)

Fibrosis is an excessive wound healing response thatoccurs in most forms of chronic liver disease andresults in the deposition of scar tissue, i.e., excess

extracellular matrix (ECM). With ongoing liver damage,fibrosis may progress to cirrhosis, which is characterizedby a distortion of the liver vasculature and architecture,and which is the major determinant of morbidity andmortality in patients with liver disease, predisposing toliver failure and primary liver cancer.1 Causal treatment,e.g., antiviral therapy for chronic hepatitis B and C, orweight loss and exercise for nonalcoholic steatohepatitis

(NASH), can retard or prevent liver fibrosis progression.However, many patients either do not respond to causaltreatment or are diagnosed with advanced fibrosis or cir-rhosis. Because fibrosis and especially cirrhosis are themajor predictors of liver-related morbidity and mortality,there is an urgent need to develop, test, and monitorantifibrotic treatments that can prevent, halt, or even re-verse liver fibrosis or cirrhosis. Although we have madeimpressive progress in our understanding of the mecha-nisms that underlie the pathogenesis of liver fibrosis in thepast two decades, translation of this knowledge into anti-fibrotic therapies has ground to a halt short of clinicaltrials. In this review we will focus on current challengesand pitfalls in the development of antifibrotic drugs, andon strategies to overcome the obstacles toward their clin-ical validation.

Promising Targets for Antifibrotic TherapiesHepatic fibrosis results from a dynamic process,

characterized by a preponderance of fibrogenesis (theexcess synthesis and deposition of ECM), over its re-moval (fibrolysis). Thus, even advanced fibrosis andpossibly cirrhosis can regress once the fibrogenic triggeris eliminated and fibrolysis prevails over fibrogenesis.1-3

The complex biology of hepatic stellate cells and myo-fibroblasts (collectively termed HSC), the major pro-ducers of excessive ECM in liver fibrogenesis, has beencovered extensively in recent reviews.4 Other profibro-genic nonparenchymal cells act upstream of activatedHSC (summarized in Table 1).

Abbreviations: BDL, bile duct ligation and scission; CCl4, carbon tetrachloride;CTGF, connective tissue growth factor; DDC, 3,5-diethoxycarbonyl-1,4-dihydro-collidine; ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition;HSC, hepatic stellate cells; HVPG, hepatic portal vein pressure gradient; MMP,matrix metalloproteinase; NASH, nonalcoholic steatohepatitis; PDGF, platelet-derived growth factor; TAA, thioacetamide; TIMP, tissue inhibitor of metallopro-teinases

From the 1Division of Gastroenterology and Hepatology, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA.

Received July 2, 2008; accepted June 7, 2009.Supported by an appointment grant by the Beth Israel Deaconess Medical Center,

and the National Institutes of Health (grants NIH 1 R21 DK076873-01A1 andNIH U19 AI066313 RFA “Hepatitis C Cooperative Research Centers”) toD.S. Y.P. was the recipient of a Sheila Sherlock fellowship of the European Associ-ation for the Study of the Liver.

Address reprint requests to: Detlef Schuppan, M.D., Ph.D., Division of Gastro-enterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Med-ical School, Dana 501, 330 Brookline Avenue, Boston, MA 02215. E-mail:[email protected]; fax: 617-667-2767.

Copyright © 2009 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.23123Potential conflict of interest: Nothing to report.

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Targets for antifibrotic therapies are cells, signalingpathways, and molecules critical for fibrosis progres-sion or reversal. They can therefore address (1) HSCactivation and recruitment, (2) activation of cells up-stream of HSC activation, (3) profibrogenic growthfactors, cytokines and other mediators, (4) intracellularprofibrogenic pathways in HSC and cells upstream oftheir activation, and (5) stimulation of fibrolytic pro-cesses to reverse existing fibrosis. Recently it has be-come evident that activated HSC (myofibroblasts) canalso originate from periportal or perivascular fibro-blasts,5,6 bone marrow-derived circulating fibrocytes7,8

that are recruited from the bloodstream during chronic

liver injury, or from liver epithelia by way of the pro-cess of epithelial-to-mesenchymal transition (EMT)9,10

(Fig. 1). However, fibrocytes and EMT contribute nomore that 5%-10% to the myofibroblast population inmost experimental studies. A prominent role of acti-vated cholangiocytes, i.e., epithelial cells of smallductular proliferations that are related to progenitorcells, in fibrogenesis has only recently become evident.They are a universal finding in liver fibrosis and theirproliferation correlates with progression of chronicliver disease of virtually any etiology.11,12

Activated cholangiocytes secrete fibrogenic growth fac-tors such as transforming growth factor beta (TGF�)1,

Table 1. Cellular Targets Other than HSC/MF Involved in Liver Fibrogenesis

Cells Model Progression Reversal References

Macrophages CCL4 Yes Yes 111B cells CCL4 Yes ? 112NK cells DDC, CCL4 Yes ? 113T cells (CD8�) CCL4 Yes ? 114Fibrocytes BDL Yes ? 7Epithelial-to-mesenchymal transition (EMT) CCL4, BDL Yes ? 10, 27Bone marrow-derived CCL4, TAA Yes Yes 8, 115

Fig. 1. The myofibroblast as key profibrogenic effector cell in the liver. Activated hepatic stellate cells/myofibroblasts are a heterogeneous cellpopulation arising from transdifferentiation of quiescent HSC, liver fibroblasts, and to a lesser degree from activated/injured liver epithelia by wayof EMT and from bone-marrow-derived circulating fibrocytes. These myofibroblasts are characterized by increased proliferation, migration, andcontractility, and a relative resistance to apoptosis. Apart from up-regulating the synthesis and deposition of various ECM components, fibrolysis isfurther compromised by way of an increased synthesis of TIMP-1 and a decreased production of fibrolytic MMPs. Other cell types and various stimulitrigger or maintain active fibrogenesis. On the other hand, once these triggers subside and with the help of antifibrotic agents, fibrosis can be resolvedby way of proteolytic removal of excess ECM, often by the same cells that play a central role in fibrogenesis, such as HSC and macrophages/Kupffercells.

HEPATOLOGY, Vol. 50, No. 4, 2009 POPOV AND SCHUPPAN 1295

TGF�2, connective tissue growth factor (CTGF), andplatelet-derived growth factor (PDGF)-BB that drive theactivation of HSC,13-16 de novo express profibrogenic in-tegrin �v�617, produce ECM components such as base-ment membrane proteins,18 and induce early programs ofductal plate formation by releasing Hedgehog ligands thatstimulate HSC.19 Activated HSC, in turn, producegrowth and survival factors for adjacent cholangiocytes,creating a positive fibrogenic feedback loop (Fig. 2).

Preclinical Models to Test PotentialAntifibroticsIn Vitro Models

In vitro models are necessary to advance our under-standing of the molecular pathogenesis of liver fibrosis.4

They also permit high-throughput testing and improve-ment of potential antifibrotic agents, mainly due to lowcost and high reproducibility. However, the usefulness ofin vitro models, such as culture-activated HSC and HSClines for drug discovery is limited, because they do notreflect the complex interactions that orchestrate fibrogen-esis or fibrolysis in vivo, such as (1) contributions of, orinteractions with, other cell types; (2) the many cytokines,growth factors, and other mediators that are produced byother cells; (3) the normal or altered ECM; and (4)changes in vascular architecture, oxygen supply, and pro-duction of reactive oxygen species.

Culture-Activated HSC and Hepatic Stellate CellLines. Although activated HSC are undoubtedly the ma-jor producers of the excessive and abnormal ECM in liverfibrosis, these cells are heterogeneous in vivo, and mayderive from different precursors, including portal orperivenular fibroblasts,20 circulating fibrocytes,7,21 orfrom epithelial-mesenchymal transition (EMT)9,10 (Fig.1).

When cultured on plastic, freshly isolated quiescent rator human HSC undergo spontaneous fibrogenic activa-tion to a myofibroblast-like cell. This phenomenon hasbeen widely used to study basic mechanisms of HSC ac-

tivation and to characterize potential antifibrotic agents.4

However, recent studies suggest a note of caution in usingthis model, because significant phenotypical differenceswere found between in vitro and in vivo activated HSC.Comparison of global gene expression patterns of culture-activated HSC with activated HSC freshly isolated fromfibrotic liver revealed an overlap of only 56%-63% ofgenes, with a better approximation when culture activatedHSC were cocultured with Kupffer cells.22 Several HSClines have been generated and characterized, such asclones of spontaneously immortalized rat HSC isolatedfrom CCL4-fibrotic liver,23 human LX-1 and LX-2 HSCimmortalized by SV40 T-antigen,24 or TWNT, a humanHSC line generated by retroviral transfection of the te-lomerase reverse transcriptase gene.25 Despite their limi-tations, these cell lines are valuable and cost-efficient toolsin early drug discovery.

Culture of Other Cell Types Implicated in HepaticFibrogenesis. Activated cholangiocytes play an impor-tant role in fibrogenesis.16,17,26 Their inhibition preventsexcessive release or activation of profibrogenic growth fac-tors, such as TGF�1, TGF�2, or CTGF. Cell lines ofactivated cholangiocytes have become available for testingof inhibitors.17,27,28 Cultures of hepatocytes, Kupffercells, or various lymphocyte subpopulations are currentlyof limited utility, because these cells can display both fi-brogenic or fibrolytic activities, depending on context andstate of activation.3,29

Precision-cut Tissue Culture Slices. Freshly pre-pared precision-cut liver slices are able to maintain hepa-tocyte metabolic functions for at least 24 hours in vitro,which is useful for toxicology research. Recently, culturedslices from normal and fibrotic liver were suggested as atool to study fibrogenesis30 and utilized to test potentialantifibrotic agents such as pentoxiphylline and the ty-rosine kinase inhibitor Gleevec.31 Although the produc-tion of standardized organ slices is not trivial, this methodoffers advantages over conventional cell culture. First,slices can be prepared directly from fibrotic liver, which

Fig. 2. Profibrogenic crosstalk between activatedcholangiocytes and myofibroblasts. Activated, prolifer-ating cholangiocytes (“ductular reaction”) produce avariety of fibrogenic stimuli acting on HSC and myofi-broblasts, including cytokines (TGF�2�TGF�1),growth factors (PDGF-BB, CTGF), bioactive molecules(ET-A), and survival/differentiation factors (Hedgehogligands, HhL). Activated cholangiocytes also expressintegrin �v�6 that binds and activates TGF�1, thusdriving local fibrogenesis. In a positive feedback loop,activated HSC produce growth and survival factors(HGF, bFGF, IL-6, HhL) for cholangiocytes, which rep-licates an embryonic program of ductal plate formationand further amplifies the fibrotic response.

1296 POPOV AND SCHUPPAN HEPATOLOGY, October 2009

will encompass the naturally occurring spectrum of acti-vated HSC. Second, they contain the hepatic (parenchy-mal and nonparenchymal) cells in their authentic three-dimensional (3D) fibrotic microenvironment, whichlargely eliminates in vitro culture artifacts and especiallythe concern that effects due to non-HSC targeting areoverlooked. Third, multiple drugs can be screened effi-ciently in slices prepared from just a few animals, which isan important advantage in view of growing ethical con-cerns over the use of large numbers of laboratory animals.Fourth, this system permits parallel testing of hepatoxicityof the tested drugs. However, more studies are needed tovalidate the organ slice culture model for routine antifi-brotic drug screening.

Artificial Organ Tissue Culture Engineering.Rapid progress has been made in micro- and nanotech-nologies that aim at restoring the normal or pathologicalliver architecture in a precisely controlled environment byway of bioengineering. Miniaturized, multiwell culturesystems for human liver cells with optimized microscalearchitecture that maintain phenotypic functions of hepa-tocytes for several weeks have been engineered.32 Thispioneering work at the interface of physics, engineering,and biology has changed traditional 2D cell culture prac-tices to more physiological 3D matrices, with the poten-tial to reconstruct the multicellular hepatic environmentin vitro. Thus, a controlled 3D coculture system usinghepatocytes and 3T3 fibroblasts was recently reported.33

Projects to create multicellular artificial cocultures ofhepatocytes, HSC, endothelial cells, Kupffer cells, andcholangiocytes are under way and may provide a highlyreproducible and cost-efficient platform to develop andtest antifibrotic drugs in a nearly physiological hepaticmicroenvironment.

Animal ModelsSmall animal models remain indispensable to (1) study

basic mechanisms of fibrosis progression or reversal, (2)

confirm relevant targets, and (3) prove the efficacy ofantifibrotic therapies. Because their characteristics havebeen reviewed recently,3 we will only highlight the use ofsmall animal models from the perspective of antifibroticdrug development. Mouse models are increasingly pre-ferred over traditional rat models because they (i) allowmore cost-effective studies, (ii) require lower amounts ofagents (which can be a limiting factor in early phases ofdrug development), and (iii) permit thorough target val-idation using genetically modified (transgenic or gene-deleted) animals. Currently available mouse models aresummarized in Table 2.

Interpretation of Results from Animal Models. It isgenerally accepted that a promising drug will have toshow efficacy in at least two mechanistically distinct fibro-sis models to exclude “model-specific” artifacts. Addition-ally, the minimal quality requirements for satisfactorypreclinical testing are (1) the use of sufficient numbers ofanimals to overcome individual heterogeneity in fibrosisprogression (n � 8-15 per group depending on the mod-el); (2) analysis of liver samples of sufficient size (5-10% ofthe liver) to eliminate considerable sampling variability;and (3) use of complementary quantitative and semiquan-titative parameters to determine fibrosis, fibrogenesis, andfibrolysis by way of: (i) total collagen as measured byhydroxyproline content. (ii) morphometrical connectivetissue assessment (Sirius Red staining); (iii) liver architec-ture as assessed by semiquantitative scoring; (iv) quanti-tative reverse-transcription polymerase chain reaction(RT-PCR) for transcripts related to fibrogenesis and fi-brolysis; and (v) proteolytic activities. Specific attentionneeds to be paid to the genetic background of the mice,which influences their susceptibility to develop liver fibro-sis. Therefore, the choice of a susceptible strain for fibrosisstudies, the purity of the genetic background, and the useof wildtype littermates as controls are central for the in-terpretation of results.

Table 2. Overview of Rodent Models of Liver Fibrosis

Name Type Type of Fibrosis Species References

CCL4 Hepatotoxin Panlobular Rat, mouse 36, multipleBile duct ligation (BDL) Biliary obstruction Biliary Rat, mouse 17, MultipleMdr2–/– Knockout Biliary Mouse 343,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) Hepatotoxin Biliary Mouse 35Thioacetamide (TAA) Hepatotoxin Panlobular Rat, mouse 59, MultipleMethionine- and choline-deficient diet (MCD) NASH Panlobular Rat, mouse 116MCD�high fat in OLEFT rats NASH, insulin resistance Panlobular Rat 40SREBP-1c-TG NASH Panlobular Mouse 39L-SACC1 mice�high fat NASH Panlobular Mouse 38TGF�1-TG Transgenic, conditional Panlobuar Mouse 42PDGF-B-TG Transgenic, conditional Panlobular Mouse 43PDGF-C-TG Transgenic Panlobular Mouse 41


Animal Models of Biliary Fibrosis. The model ofsecondary biliary fibrosis due to bile duct ligation andscission (BDL) in rats is widely accepted, technically sim-ple, and applicable also to mice. It results in a reproduc-ible portal tract fibrosis over a short period of time (4-6weeks in rats, 2-3 weeks in mice). However, it shows littleresemblance to human fibrotic liver diseases (except forchronic biliary obstruction) and an altered metabolism ofmost drugs that are primarily secreted through the biliarytract or that undergo enterohepatic circulation. There-fore, mice that lack the hepatocyte phospholipid flippaseMdr2 (abcb4)�/�, the mouse homolog of the humanMdr3 gene) are increasingly used. They develop biliaryfibrosis due to cholangiocyte proliferation and massiveup-regulation of profibrogenic genes as early as 4 weekspostpartum.34 Fibrosis progression is spontaneous (i.e.,does not require manipulation or toxin administration),progressive, and highly reproducible. Furthermore, it re-sembles human primary sclerosing cholangitis, whilemaintaining features common to other advanced chronicliver diseases including the development of liver cancer at8-10 months of age.

When mice are fed 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) for 6-8 weeks, they develop pronouncedaberrant ductular proliferations, similar to Mdr2�/�mice. The model was recently characterized in severalmouse strains,35 and is especially attractive because it canbe applied in genetically modified mice.

Hepatotoxin-Induced Animal Models of Liver Fi-brosis. Liver fibrosis induced by carbon tetrachloride(CCl4) results from repetitive (necrotic) hepatocyte deathand continues to be popular because it allows the study ofacute liver injury, advanced fibrosis, and fibrosis reversal(reviewed in Ref. 3). Although mice generally show lesstoxin-induced fibrosis than rats, the different susceptibil-ity of inbred strains to CCl4-induced liver fibrosis is wellcharacterized.36 The model’s major disadvantages are thesevere hepatocyte necrosis and its dependence on massiveoxidative stress that is not found to such an extent inhuman chronic liver diseases. A similar degree of fibrosiscan be achieved by chronic injection of the hepatotoxinthioacetamide (TAA). Although it is essentially impossi-ble to separate antiinflammatory or free radical scaveng-ing activities of drugs from antifibrotic effects when theseare given during toxin administration, the toxin modelsare very useful to test the efficacy of agents to speed upfibrosis reversal after toxin administration has been dis-continued. Furthermore, the pattern of fibrosis achievedclosely resembles the panlobular and parenchymal fibrosisthat is found in most human chronic liver diseases.

Animal Models of NASH. More recently, severalmodels have been described that mimic important aspects

of human NASH and fibrosis, in combination with fea-tures of the metabolic syndrome.37-39 The most efficientmodels exploit a genetic predisposition for the metabolicsyndrome in combination with a high-fat diet. When amethionine choline-deficient diet was added as a third“hit,” obese diabetic Otsuka Long-Evans TokushimaFatty (OLETF) rats developed cirrhosis within 8 weeks.40

Transgenic and Gene Deletion In Vivo Models.Genetic models have great value to confirm factors andmechanisms that drive fibrogenesis or fibrolysis in vivo,even more because the models have been refined with theuse of tissue- and cell-specific, or conditional transgenesor deletions. An example are transgenic mice with consti-tutive overexpression of PDGF-C,41 or with conditionaloverexpression of TGF-�42 or PDGF-B.43 However,these models need further refinement because expressionof the profibrogenic factors is typically driven by way ofthe albumin promoter in hepatocytes, whereas physiolog-ically they are mainly produced by inflammatory cells andactivated HSC.

Antifibrotic Drug DevelopmentImpressive progress in our understanding of molecular

mechanisms of fibrosis progression uncovered severalpromising molecular targets for antifibrotic treatments(Table 3) (for reviews, see Schuppan and Afdhal,1 Fried-man,5 Bataller and Brenner,6 and Rockey44). However,transfer of these experimental treatments to clinical prac-tice has been slow.

A major obstacle to antifibrotic drug development hasbeen the usually slow progression of liver fibrosis in hu-mans, coupled with a lack of sensitive and noninvasivemeans to assess fibrosis or fibrogenesis (see below). Fur-thermore, many patients are asymptomatic even whenfibrosis is already advanced, or present with decompen-sated cirrhosis, which would require not only inhibitionbut also reversal of established fibrosis. Accordingly, anti-fibrotic drug development must consider the followingtwo scenarios, balanced against the risk, cost, ease, andduration of treatment: (1) long-term: mainly inhibitingfurther progression, using a low-risk, relatively cheap oralagent; (2) short-term/interval: inducing reversal of (de-compensated) disease or incipient cirrhosis, using a po-tentially high risk, parenteral, and possibly expensiveagent.

Targeted Approaches. Targeted approaches are di-rected to known molecular targets or pathways that arecritically involved in fibrogenesis or fibrolysis, and that donot overlap significantly with unrelated pathways (whichwould incur unwanted side effects). Such agents can befunction-blocking antibodies, or preferably small mole-cule inhibitors. A limitation of the antibody approach is


its restriction to cell surface or extracellular targets and anoften low bioavailability at the target site. However, theymay allow rapid proof of principle testing in vivo. Smallmolecules are usually obtained by way of screening ofexisting compound libraries in a cellular readout system,followed by further chemical refinement. This process iseffort- and time-consuming, but may yield orally avail-able drugs. However, most of these developments neverreach clinical phase II-III studies due to unwanted (off-site) side effects.

Bioinformatics is gaining importance in drug develop-ment and improvement, especially as in silico drug engi-neering and predictive activity modeling.45 This approachis based on 3-10 compounds with known activity towarda target. Affinities of these compounds have to be known,but an in-depth understanding of drug-target interactionsis not required. From a virtual library of several millioncompounds, structure-affinity comparisons and in silicooptimization will yield novel lead compounds that aresynthesized and tested in a molecular or cellular readoutassay for affinity, specificity, and other desired properties.Subsequently, these improved compounds will then bereentered into a novel cycle of in silico optimization andtesting.

Several targeted approaches have been directed towardthe profibrogenic TGF� signaling pathway, e.g., using asoluble TGF� receptor type II,46 TGF� blocking anti-bodies, TGF� antisense oligonucleotides, or moleculesthat interfere with downstream (smad-mediated) signaltransduction. Two monoclonal antibodies againstTGF�1, Lerdelimumab and Meselimumab, are currentlyin clinical phase I-III trials in pulmonary fibrosis, systemicsclerosis, and postoperative scarring in glaucoma pa-tients.47 There is an important limitation in targeting theTGF� pathway systemically (this also applies to otherphysiologically relevant targets such as tissue inhibitor of

metalloproteinases [TIMP-1] or certain matrix metallo-proteinases [MMPs]), because apart from stimulatingwound healing and fibrosis, TGF� is also a central inhib-itor of uncontrolled inflammation, and essential in induc-ing epithelial differentiation and in triggering apoptosis.This raises safety concerns for the general and long-termuse of TGF� inhibition, especially in patients withchronic (hepatic) inflammation. Another yet preclinicalstrategy is to block TIMP-1, a major effector of liver fi-brosis, by use of a recombinant mutant protein derivedfrom its high-affinity ligand MMP-9.48 An intriguingnovel target is the integrin �v�6, a cell surface receptorthat is expressed on activated epithelia during embryogen-esis, wound healing, and tumorigenesis.49 �v�6 anchorsthese epithelia to a provisional ECM of tenascin and fi-bronectin17 and through tethering latent TGF�1 pro-motes its proteolytic activation.50 In the liver, �v�6 isexclusively expressed on activated cholangiocytes that arepotent promoters of liver fibrogenesis.17,26,51 Its successfultargeting by a blocking antibody51 or a small moleculeinhibitor specifically blocked proliferation of profibro-genic cholangiocytes and concomitant TGF�1 activationat the site of fibrogenesis.17,26 Promising targeted ap-proaches are summarized in Table 3.

Alternative and Complementary Medicine. Alter-native medicines for chronic liver disease, mostly herbalpreparations from single or multiple plants, can be tracedback 4000 years in ancient China and India and holdpromise for the development of cheap and potentiallylow-risk “natural” drugs. Numerous studies, mainly injournals that are not easily accessible, report antifibroticeffects of herbal medicines. However, there is a slowlygrowing number of several better-defined active ingredi-ents from herbal medicines that show antifibrotic proper-ties, such as baicalein, curcumin, and silymarin52,53

(Table 4). Although over-the-counter botanicals repre-

Table 3. Selection of Molecular Targets in Liver Fibrosis

Target Cells Drug References

TGF� HSC, cholangiocytes, inflammatorycells, endothelia

Soluble type II receptor, anti-TGF�antibody, anti-TGF� small moleculeantagonists

46, 47

Integrin �v�6 Activated epithelia Small molecule antagonist, blocking AB 17, 26, 51Endothelin-A receptor Cholangiocytes, HSC Small molecule antagonists 117Cannabinoid receptors CB1, CB2 Cholangiocytes, HSC Small molecule CB1 antagonistsCB2

agonists118

Toll like receptor-4 (TLR-4) Macrophages, HSC Small molecule antagonists (also todownstream targets)

119

Angiotensin-1 receptor, angiotensinconverting enzyme (AT1/ACE)

HSC Numerous small molecule inhibitors 6

Caspases Hepatocytes Small-molecule inhibitors of caspaseactivation

120

Farnesoid receptor (FXR) HSC FXR agonists 121

HSC, hepatic stellate cell; TGF, transforming growth factor.


sent a multibillion market in the US alone, significantprogress in this area is hampered by two major obstacles.First, the reliance of traditional Eastern medicine on em-piricism and a holistic philosophy, where controlled stud-ies are not considered necessary and where particularherbs are believed to be active only in certain combina-tions. This is in conflict with the Western approach,which relies on scientific evidence requiring adequatelydesigned, placebo-controlled, randomized trials to provethe efficacy of structurally defined agents rather than ofcomplex herbal mixtures.52 Second, the liberal use ofpoorly standardized crude extracts or complex mixtures ofbotanicals poses an increased risk of hepatotoxicity.54 As aresult, only a few botanicals are currently in recognizedclinical trials, including the flavonoids silymarin or silibi-nin from milk thistle for patients with chronic hepatitis Cor nonalcoholic steatohepatitis55 (see http://clinicaltrials-.gov/ for a complete list).

Drug Repositioning. Drug repositioning is the effortto develop treatments by finding novel applications fordrugs that are already in clinical use for other indica-tions.56 Examples for liver fibrosis are immunosuppres-sants such as rapamycin,57 antidiabetic glitazones,58 theantiparasitic drug halofuginone,59,60 the tyrosine kinase-targeted anticancer drugs sunitinib,61 or imatinib mesy-late (Gleevec),62 and blockers of the angiotensin system.6

Because the clinical safety and efficacy profile is alreadyknown for these agents, drug repositioning offers thechance of a “fast track” introduction of novel treatmentsinto clinical practice. However, their antifibrotic efficacyin vivo may be limited, requiring either their combinationwith other drugs, or their targeted delivery to relevantliver cells.

Targeted Drug Delivery to Fibrogenic Liver Cells.Effective targeting will increase local drug concentrationsand diminish potential side effects. By nature, activatedHSC are a major target for antifibrotic therapies, but drugtargeting to HSC has been difficult because cell surfacemolecules up-regulated during fibrogenesis and inflam-

mation, such as the mannose 6-phosphate receptor andcertain integrins, are also found on other liver cells, suchas hepatocytes, inflammatory, and endothelial cells.63 Afew exceptions, such as the PDGF� receptor or the colla-gen VI receptor, are mainly expressed on activated HSCand are virtually absent from normal liver.13,64 Thus, theimmunosuppressive and antiproliferative drug mycophe-nolate mofetil65 or apoptosis-inducing gliotoxin66 wascoupled to mannose6-phosphate (insulin-like growth fac-tor type II) receptor binding constructs that after intrave-nous injection mainly targeted HSC and sinusoidalendothelial cells. Coupling proapoptotic gliotoxin to asingle-chain antibody against synaptophysin, which is ex-pressed in activated HSC-induced apoptosis of activatedHSC, and thus reduced fibrosis in the CCl4 model.67

Alternatively, vitamin A-modified liposomes increaseddelivery of small interfering RNA (siRNA) directedagainst the putative collagen-specific chaperone HSP47to vitamin A-storing HSC, causing a pronounced antifi-brotic effect in the CCl4 and BDL-induced liver fibrosis.68

Once effective targeted delivery of siRNA to activatedHSC is confirmed, the versatility of this platform holdspromise, because it permits the silencing of any knownprofibrogenic gene. Recently, targeting of adenovirus tothe PDGF� receptor using the receptor binding octapep-tide (CSRNLIDC) cloned upstream of a single-chain an-tibody fragment directed against the adenoviral knob wasemployed to redirect adenoviral delivery of siRNA fromhepatocytes to activated HSC.69

Noninvasive Means to Assess Liver Fibrosis andFibrogenesis

The major obstacle to antifibrotic drug developmenthas been the difficulty in defining accepted surrogate end-points for clinical trials. A slowly evolving disease (manyyears to several decades for the development of cirrhosis,hepatic decompensation, or hepatocellular carcinoma de-velopment) or an elusive endpoint (liver biopsy) repre-sents a significant hurdle for study design. Notably, mere

Table 4. Potential Antifibrotics Derived from Complementary and Alternative Medicine

Active Compound Source Mode of Action References

Silibinin-1/-2(Silymarin) Milk thistle Antioxidant, TGF�-inhibition, hepatitis C viral suppression 52, 55, 122

Baicalein TJ-9 (Sho-saiko-to) Antioxidant, inhibition of HSC proliferation 123

Curcumin TurmericAntioxidant, NF-kB and TNF�-inhibition, inhibition of HSC

proliferation, induction of HSC apoptosis 124Emodin TJ-135 PDGF-�R inhibition 125Salvianolic acid,

tanshinones Salvia miltiorrhiza Antioxidant, TGF�-inhibition 126Berberin Coptis Antioxidant, activation of superoxide dismutase 127Trans-resveratrol Red wine Antioxidant, NF-�B inhibition 128

HSC, hepatic stellate cell; NF-�B, nuclear factor kappa B; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor.


inhibition of fibrosis progression (i.e., without the above-mentioned hard endpoints) is only slowly becoming ac-cepted by regulatory authorities, such as the Food andDrug Administration (FDA).

To date, clinical studies on the progression or regres-sion of liver fibrosis, with or without potential antifibrotictreatment, have relied on sequential liver biopsies andassessment of fibrosis progression using the conventionalstaging systems. Liver biopsy is invasive and risky,70 sam-ples only 1/50,000 of the liver, and is prone to consider-able sampling error. This is illustrated by a study thatcompared laparoscopic liver biopsies from the right andthe left lobe in patients with hepatitis C. One-third ofpaired samples differed by at least one out of the fourMetavir stages, and 14.5% showed advanced fibrosis inone and cirrhosis in the other lobe (stage F3 versus stageF4).71 Construction of smaller virtual biopsies from sur-gical liver biopsies confirmed that in hepatitis C even largebiopsy cores (�25 mm) confer an inherent one-stagesampling variability of 25%.72 Even higher sampling vari-abilities are reported for NASH and biliary fibrosis.73,74

Therefore, the following requirements must be fulfilledfor clinical studies that use conventional staging of liverbiopsy alone in order to prove a significant antifibroticdrug effect: (1) at least 200-500 well-selected patientswith a single etiology of their liver disease; (2) patientswith an intermediate stage of fibrosis, preferably MetavirF2, which serves as the best predictor for further fibrosisprogression while still permitting a sensitive detection ofdrug-induced changes; and (3) a study duration of at least2-5 years, with pre- and poststudy biopsies. Thus, basedon staging alone, even exploratory phase I and II studiesare hampered by the need for a significant sample size andby a high risk of failure. However, even with currentlyavailable techniques, patient numbers and study durationmay be reduced significantly, pending prospective valida-tion against hard endpoints. Thus, conventional biopsyreadouts can be improved, such as by semiquantificationof activated myofibroblasts by way of staining for �-SMAor TGF�,75 or by quantitative PCR quantification oftranscripts that are related to fibrogenesis or fibrolysis.76

When assessing progression with or without therapy, acohort of, for example, HCV-infected patients with inter-mediate fibrosis (Metavir stage 2) would be preferable,because this would best allow detection of modest changesin fibrous tissue in a population with a high likelihood ofprogression. Antifibrotic effects will be more difficult todetect in patients with precirrhotic or cirrhotic disease,because collagen content increases exponentially witheach stage, resulting in a roughly 4 to 5-fold increase inliver collagen in stage 4 (cirrhosis) over stage 2.72 On theother hand, patients with cirrhosis obviously need an an-

tifibrotic therapy most urgently. In these patients, a func-tional parameter that is correlated with the extent ofremodeling and fibrosis, such as hepatic portal vein pres-sure gradient (HVPG) is plausible.77 In fact, a recentstudy confirmed HVPG as the best predictor of mortalityin cirrhotic patients with compensated Child’s A cirrho-sis.78

Yet the shortcomings of traditional study design andtechnologies highlight the need for the identification ofnew biomarkers or imaging techniques that allow an exactassessment of the degree of fibrosis and, more important,of the dynamic processes of fibrogenesis or fibrolysis, inorder to predict fibrosis progression or to monitor theeffect of antifibrotic therapies.

Serum Fibrosis Markers. The recent literature is re-plete with studies that employ serological fibrosis markersand their combinations to cross-sectionally stage liver fi-brosis (reviewed in 1, 79-81). These have mainly beenvalidated in chronic hepatitis C and can show diagnosticaccuracies around 80% for the differentiation between noor mild (Metavir F0/1) and moderate to severe (MetavirF2-4) fibrosis. Their inability to differentiate intermedi-ate fibrosis stages, e.g., stage 1 from 2, precludes their useas surrogates in clinical studies with potential antifibrot-ics, a setting in which fibrosis progression is slow. Further-more, these markers are only used for a dichotomousdifferentiation of patients into two crude (no/mild vs.moderate/severe) fibrosis categories, whereas fibrosis pro-gression is nonlinear.82,83 Furthermore, even when usingthe dichotomous classification there is a high indetermi-nate rate with an inability to stage fibrosis in 30%-70% ofpatients with intermediate fibrosis stages. To complicatematters, the amount of hepatic fibrous tissue increasesexponentially with every fibrosis stage,72 whereas thesesurrogates are continuous variables. Finally, for their val-idation these markers and algorithms have to be com-pared to liver biopsy, which, as mentioned above, is farfrom an ideal “gold standard” due to inherent samplingvariability. Given the drawbacks of liver biopsy, a recentstudy calculated the chance to identify a given markerpanel to correctly stage fibrosis according to the above-mentioned criteria.84 By assuming the realistic scenario of90% sensitivity and 80% specificity of liver biopsy tocorrectly identify significant fibrosis F � 2, even a perfectserum test (accuracy 99%) would result in a test accuracybelow 80%, well in the range of current marker panels.The fibrosis markers and their combinations have almostexclusively been validated cross-sectionally, i.e., in com-parison to fibrosis stage, whereas those markers that arerelated to matrix metabolism may rather reflect dynamicalterations of connective tissue turnover, i.e., fibrogenesisor fibrolysis. Thus, several studies suggest that certain


marker combinations, such as Fibrotest, the enhancedliver fibrosis panel of ECM markers (ELF), or other se-lected ECM markers may have more value in predictingprogression rather than fibrosis stage.85-90 However, theirvalidation as dynamic markers is difficult and will requiremore long-term studies. Such studies should also be com-plemented by determination of hepatic fibrogenesis byquantitative PCR for transcripts that are related to fibro-genesis or fibrolysis in matched liver biopsies.

Attempts to Discover Better Serum Markers UsingProteomics and Transcriptomics. Molecular alter-ations associated with liver fibrogenesis or fibrolysis ofteninvolve secreted proteins, making it likely that specificbiomarkers for different phases of the disease do exist inserum. Proteomic technologies based on protein fraction-ation and mass spectrometry (MS), with the aid of bioin-formatics, provide the means to compare protein profilesin normal and pathological serum. Serum is depleted ofup to 20 of the most abundant proteins, resulting in �2%of the original protein content but �99% of originalprotein variety. Depleted sera are then either separated by2D gel electrophoresis (DIGE) that allows comparativeanalysis of three different samples in a single gel, or bytryptic digests of serum proteome fractions that have beenlabeled with so-called isobaric tags (iTRAQ technology),permitting parallel analysis of up to eight serum sam-ples.91-93 Identification of the in gel trypsin digested pro-teins (DIGE) or of the tryptic iTRAQ peptides is done byMS. The power of these technologies lies in a quantitativecomparison of an identified peptide or protein in three(DIGE) or eight (iTRAQ) serum samples. However, theirsensitivity to detect proteins at the lower end of serumconcentrations, i.e., in the picomolar range, such as cyto-kines or ECM peptides or proteins, is still insufficient.Furthermore, differential glycosylation generates com-plex patterns for a single protein in DIGE, which is basedon charge separation in the first dimension.

Nonetheless, projects that employ proteomics are onthe way, not only in serum or plasma samples that can becorrelated to readouts of matched biopsies, but also inrodent models of fibrosis progression or reversal. The lat-ter approach is promising, because mechanisms and mol-ecules in fibrogenesis and fibrolysis in rodents are highlysimilar to those in humans, and representative liver tissuecan be sampled for correlation to the extent of hepaticfibrogenesis or fibrolysis. This allows the transfer of thenewly discovered biomarkers rapidly to the human sys-tem.

Novel serum markers may also be found by analysis ofthe liver transcriptome, an approach which has been at-tempted mainly in biopsies from patients with hepatitisC.94,95 This technology is far simpler than serum pro-

teomics and may detect low abundancy gene expression.However, most of the regulated gene products will not beshed into the circulation and are unspecific or unrelated tothe fibrogenic process, requiring a high effort to find andvalidate a single novel serum marker.

Imaging of Liver Fibrosis. Imaging of liver fibrosis,and in particular hepatic fibrogenesis, with conventionalmethodology has been elusive. Ultrasonography, com-puterized tomography, magnetic resonance imaging(MRI), positron emission tomography (PET), and singlephoton emission computerized tomography (SPECT) arenot able to detect fibrosis and are not even sensitiveenough to diagnose cirrhosis in many patients.1 Diffu-sion-weighted MRI may offer some differentiation, butwill likely not be able to exactly stage fibrosis.96 MR tex-ture analysis and double-contrast MRI using suprapara-magnetic iron and gadolinium as contrast agents mayyield a better fibrosis assessment but are in their infan-cy.97,98 Recently, a functional MRI method was proposedthat is based on measuring hemodynamic response in theliver to hypercapnia and hyperoxia. Increase in liver per-fusion was attenuated according to degree of fibrosis inrats.99 This approach, if validated in patients with liverdisease, may offer an additional noninvasive diagnostictool for evaluation and follow-up of liver diseases.

Ultrasound Elastography (Fibroscan). This is anovel, noninvasive bedside method to assess liver fibrosisby measuring hepatic stiffness. An intercostally placedprobe transmits low amplitude shear waves through theliver, and their velocity is picked up by an integratedultrasound device (pulse-echo ultrasound). Shear wavevelocity is inversely correlated with liver stiffness. Themethod is painless and without risk, takes only 5 minutes,and can be performed in a fairly standardized way afteronly a short training period. Furthermore, it measures anovel, i.e., mechanical quality of the liver related to fibro-sis and, when compared to biopsy, samples a 100-foldlarger volume (4 � 1 cm, �1/500 of the liver). More than100 clinical studies correlating ultrasound elastographywith liver biopsy have appeared since its first descriptionin 2003,100 including meta-analyses.101-103 These show agood correlation of stiffness values with the histologicalstage of fibrosis. Area under receiver operating curves(that reflect the diagnostic precision in select populations)yielded excellent accuracy for differentiating cirrhosisfrom noncirrhosis (AUROC between 0.90 and 0.99), es-pecially for patients with chronic hepatitis C. However,with the practical but crude histological Metavir stagingsystem (with F1 � mild, periportal fibrosis, and F4 �cirrhosis), there is high overlap between the lower stages,only part of which can be attributed to biopsy samplingvariability. In addition, severe inflammation and mechan-


ical cholestasis can significantly increase hepatic stiffness,thus confounding the fibrosis readout.104-106 Nonetheless,ultrasound elastography should prove useful for stratifica-tion of patients for inclusion into treatment studies.

MR elastography is based on similar principles as ul-trasound elastography, but studies are few and most havenot included low numbers of patients. A larger generatortransmits the shear waves which are recorded with a 1.5 TMR scanner.107 Advantages are examination of the wholeliver, an even lower observer dependency, and applicabil-ity in patients with severe obesity or narrow intercostalspaces (which can preclude ultrasound elastography). Adisadvantage is the more restricted availability. Impor-tantly, acquisition times can be shortened 10-fold (20 to 2minutes) by use of echo-planar versus spin-echo se-quences, without compromise of the stiffness readout.107

A recent study in 133 patients with various liver diseasesrevealed stunning AUROC values between 0.985 and0.998 for the diagnosis of fibrosis stages F � 2, F � 3, orF � 4, and superiority of MR elastography over ultra-sound elastography or a combination of the serologicalAST over platelet ratio (APRI) combined with ultrasoundelastography.108 Hepatic inflammation and mechanicalcholestasis are expected to be confounders as with ultra-sound elastography, but studies are lacking.

Molecular Imaging of Liver Fibrosis and Fibrogen-esis. Molecular (targeted) imaging technology is basedon a high-affinity ligand for a cell surface molecule thathas been coupled to a radio- or MRI-imaging agent.109

Ideally, fibrosis imaging constructs should be of small size,to allow penetration into the interstitial tissue, which isparticularly relevant for imaging of fibrosis or fibrogen-esis. In addition, the imaging construct should be non-toxic and have a desirable plasma half-life of 10-30

minutes, to be eliminated predominantly through thekidneys. Finally, unspecific background uptake should below to yield a good signal-to-noise ratio. Attractive targetmolecules are the abundant fibrillar collagens (for fibrosismeasurement) and cell surface markers of fibrogenesissuch as integrin �v�6 on cholangiocytes and thePDGF�R on activated HSC for quantification of fibro-genesis (Fig. 3). The availability of quantitative fibrogen-esis imaging over the whole liver could enable the testingof antifibrotic agents in small numbers of patients andover short periods of time. Early data on the feasibility ofthis approach are emerging.110

ConclusionThe stage appears to be set for a highly predictive and

effective preclinical selection and testing of antifibroticagents. Their clinical validation in phase I and II studieswill be jump-started once the desired noninvasive meth-ods for their rapid and reliable testing in small numbers ofpatients have been developed.

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Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies