Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies†
Article first published online: 15 JUN 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 4, pages 1294–1306, October 2009
How to Cite
Popov, Y. and Schuppan, D. (2009), Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies. Hepatology, 50: 1294–1306. doi: 10.1002/hep.23123
Potential conflict of interest: Nothing to report.
- Issue published online: 28 SEP 2009
- Article first published online: 15 JUN 2009
- Accepted manuscript online: 15 JUN 2009 12:00AM EST
- Manuscript Accepted: 7 JUN 2009
- Manuscript Received: 2 JUL 2008
- Beth Israel Deaconess Medical Center
- National Institutes of Health “Hepatitis C Cooperative Research Centers”. Grant Numbers: NIH 1 R21 DK076873-01A1, NIH U19 AI066313 RFA
- Sheila Sherlock fellowship of the European Association for the Study of the Liver
- Top of page
- Promising Targets for Antifibrotic Therapies
- Preclinical Models to Test Potential Antifibrotics
We have made striking progress in our understanding of the biochemistry and cell biology that underlies liver fibrosis and cirrhosis, including the development of strategies and agents to prevent and reverse fibrosis. However, translation of this knowledge into clinical practice has been hampered by (1) the limitation of many in vitro and in vivo models to confirm mechanisms and to test antifibrotic agents, and (2) the lack of sensitive methodologies to quantify the degree of liver fibrosis and the dynamics of fibrosis progression or reversal in patients. Furthermore, whereas cirrhosis and subsequent decompensation are accepted hard clinical endpoints, fibrosis and fibrosis progression alone are merely plausible surrogates for future clinical deterioration. In this review we focus on an optimized strategy for preclinical antifibrotic drug development and highlight the current and future techniques that permit noninvasive assessment and quantification of liver fibrosis and fibrogenesis. The availability of such noninvasive methodologies will serve as the pacemaker for the clinical development and validation of potent antifibrotic agents. (HEPATOLOGY 2009.)
Fibrosis is an excessive wound healing response that occurs in most forms of chronic liver disease and results in the deposition of scar tissue, i.e., excess extracellular matrix (ECM). With ongoing liver damage, fibrosis may progress to cirrhosis, which is characterized by a distortion of the liver vasculature and architecture, and which is the major determinant of morbidity and mortality in patients with liver disease, predisposing to liver failure and primary liver cancer.1 Causal treatment, e.g., antiviral therapy for chronic hepatitis B and C, or weight loss and exercise for nonalcoholic steatohepatitis (NASH), can retard or prevent liver fibrosis progression. However, many patients either do not respond to causal treatment or are diagnosed with advanced fibrosis or cirrhosis. Because fibrosis and especially cirrhosis are the major predictors of liver-related morbidity and mortality, there is an urgent need to develop, test, and monitor antifibrotic treatments that can prevent, halt, or even reverse liver fibrosis or cirrhosis. Although we have made impressive progress in our understanding of the mechanisms that underlie the pathogenesis of liver fibrosis in the past two decades, translation of this knowledge into antifibrotic therapies has ground to a halt short of clinical trials. In this review we will focus on current challenges and pitfalls in the development of antifibrotic drugs, and on strategies to overcome the obstacles toward their clinical validation.
Promising Targets for Antifibrotic Therapies
- Top of page
- Promising Targets for Antifibrotic Therapies
- Preclinical Models to Test Potential Antifibrotics
Hepatic fibrosis results from a dynamic process, characterized by a preponderance of fibrogenesis (the excess synthesis and deposition of ECM), over its removal (fibrolysis). Thus, even advanced fibrosis and possibly cirrhosis can regress once the fibrogenic trigger is eliminated and fibrolysis prevails over fibrogenesis.1–3 The complex biology of hepatic stellate cells and myofibroblasts (collectively termed HSC), the major producers of excessive ECM in liver fibrogenesis, has been covered extensively in recent reviews.4 Other profibrogenic nonparenchymal cells act upstream of activated HSC (summarized in Table 1).
|NK cells||DDC, CCL4||Yes||?||113|
|T cells (CD8+)||CCL4||Yes||?||114|
|Epithelial-to-mesenchymal transition (EMT)||CCL4, BDL||Yes||?||10, 27|
|Bone marrow-derived||CCL4, TAA||Yes||Yes||8, 115|
Targets for antifibrotic therapies are cells, signaling pathways, and molecules critical for fibrosis progression or reversal. They can therefore address (1) HSC activation and recruitment, (2) activation of cells upstream of HSC activation, (3) profibrogenic growth factors, cytokines and other mediators, (4) intracellular profibrogenic pathways in HSC and cells upstream of their activation, and (5) stimulation of fibrolytic processes to reverse existing fibrosis. Recently it has become evident that activated HSC (myofibroblasts) can also originate from periportal or perivascular fibroblasts,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 process of epithelial-to-mesenchymal transition (EMT)9, 10 (Fig. 1). However, fibrocytes and EMT contribute no more that 5%-10% to the myofibroblast population in most experimental studies. A prominent role of activated cholangiocytes, i.e., epithelial cells of small ductular proliferations that are related to progenitor cells, in fibrogenesis has only recently become evident. They are a universal finding in liver fibrosis and their proliferation correlates with progression of chronic liver disease of virtually any etiology.11, 12
Activated cholangiocytes secrete fibrogenic growth factors such as transforming growth factor beta (TGFβ)1, TGFβ2, connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF)-BB that drive the activation of HSC,13–16 de novo express profibrogenic integrin αvβ617, produce ECM components such as basement membrane proteins,18 and induce early programs of ductal plate formation by releasing Hedgehog ligands that stimulate HSC.19 Activated HSC, in turn, produce growth and survival factors for adjacent cholangiocytes, creating a positive fibrogenic feedback loop (Fig. 2).
Preclinical Models to Test Potential Antifibrotics
- Top of page
- Promising Targets for Antifibrotic Therapies
- Preclinical Models to Test Potential Antifibrotics
In Vitro Models
In vitro models are necessary to advance our understanding of the molecular pathogenesis of liver fibrosis.4 They also permit high-throughput testing and improvement of potential antifibrotic agents, mainly due to low cost and high reproducibility. However, the usefulness of in vitro models, such as culture-activated HSC and HSC lines for drug discovery is limited, because they do not reflect the complex interactions that orchestrate fibrogenesis or fibrolysis in vivo, such as (1) contributions of, or interactions with, other cell types; (2) the many cytokines, growth factors, and other mediators that are produced by other cells; (3) the normal or altered ECM; and (4) changes in vascular architecture, oxygen supply, and production of reactive oxygen species.
Culture-Activated HSC and Hepatic Stellate Cell Lines.
Although activated HSC are undoubtedly the major producers of the excessive and abnormal ECM in liver fibrosis, these cells are heterogeneous in vivo, and may derive from different precursors, including portal or perivenular fibroblasts,20 circulating fibrocytes,7, 21 or from epithelial-mesenchymal transition (EMT)9, 10 (Fig. 1).
When cultured on plastic, freshly isolated quiescent rat or human HSC undergo spontaneous fibrogenic activation to a myofibroblast-like cell. This phenomenon has been widely used to study basic mechanisms of HSC activation and to characterize potential antifibrotic agents.4 However, recent studies suggest a note of caution in using this model, because significant phenotypical differences were found between in vitro and in vivo activated HSC. Comparison of global gene expression patterns of culture-activated HSC with activated HSC freshly isolated from fibrotic liver revealed an overlap of only 56%-63% of genes, with a better approximation when culture activated HSC were cocultured with Kupffer cells.22 Several HSC lines have been generated and characterized, such as clones of spontaneously immortalized rat HSC isolated from CCL4-fibrotic liver,23 human LX-1 and LX-2 HSC immortalized by SV40 T-antigen,24 or TWNT, a human HSC line generated by retroviral transfection of the telomerase reverse transcriptase gene.25 Despite their limitations, these cell lines are valuable and cost-efficient tools in early drug discovery.
Culture of Other Cell Types Implicated in Hepatic Fibrogenesis.
Activated cholangiocytes play an important role in fibrogenesis.16, 17, 26 Their inhibition prevents excessive release or activation of profibrogenic growth factors, such as TGFβ1, TGFβ2, or CTGF. Cell lines of activated cholangiocytes have become available for testing of inhibitors.17, 27, 28 Cultures of hepatocytes, Kupffer cells, or various lymphocyte subpopulations are currently of limited utility, because these cells can display both fibrogenic or fibrolytic activities, depending on context and state of activation.3, 29
Precision-cut Tissue Culture Slices.
Freshly prepared precision-cut liver slices are able to maintain hepatocyte metabolic functions for at least 24 hours in vitro, which is useful for toxicology research. Recently, cultured slices from normal and fibrotic liver were suggested as a tool to study fibrogenesis30 and utilized to test potential antifibrotic agents such as pentoxiphylline and the tyrosine kinase inhibitor Gleevec.31 Although the production of standardized organ slices is not trivial, this method offers advantages over conventional cell culture. First, slices can be prepared directly from fibrotic liver, which will encompass the naturally occurring spectrum of activated HSC. Second, they contain the hepatic (parenchymal and nonparenchymal) cells in their authentic three-dimensional (3D) fibrotic microenvironment, which largely eliminates in vitro culture artifacts and especially the concern that effects due to non-HSC targeting are overlooked. Third, multiple drugs can be screened efficiently in slices prepared from just a few animals, which is an important advantage in view of growing ethical concerns over the use of large numbers of laboratory animals. Fourth, this system permits parallel testing of hepatoxicity of the tested drugs. However, more studies are needed to validate the organ slice culture model for routine antifibrotic drug screening.
Artificial Organ Tissue Culture Engineering.
Rapid progress has been made in micro- and nanotechnologies that aim at restoring the normal or pathological liver architecture in a precisely controlled environment by way of bioengineering. Miniaturized, multiwell culture systems for human liver cells with optimized microscale architecture that maintain phenotypic functions of hepatocytes for several weeks have been engineered.32 This pioneering work at the interface of physics, engineering, and biology has changed traditional 2D cell culture practices to more physiological 3D matrices, with the potential to reconstruct the multicellular hepatic environment in vitro. Thus, a controlled 3D coculture system using hepatocytes and 3T3 fibroblasts was recently reported.33 Projects to create multicellular artificial cocultures of hepatocytes, HSC, endothelial cells, Kupffer cells, and cholangiocytes are under way and may provide a highly reproducible and cost-efficient platform to develop and test antifibrotic drugs in a nearly physiological hepatic microenvironment.
Small animal models remain indispensable to (1) study basic mechanisms of fibrosis progression or reversal, (2) confirm relevant targets, and (3) prove the efficacy of antifibrotic therapies. Because their characteristics have been reviewed recently,3 we will only highlight the use of small animal models from the perspective of antifibrotic drug development. Mouse models are increasingly preferred over traditional rat models because they (i) allow more cost-effective studies, (ii) require lower amounts of agents (which can be a limiting factor in early phases of drug development), and (iii) permit thorough target validation using genetically modified (transgenic or gene-deleted) animals. Currently available mouse models are summarized in Table 2.
|Name||Type||Type of Fibrosis||Species||References|
|CCL4||Hepatotoxin||Panlobular||Rat, mouse||36, multiple|
|Bile duct ligation (BDL)||Biliary obstruction||Biliary||Rat, mouse||17, Multiple|
|Thioacetamide (TAA)||Hepatotoxin||Panlobular||Rat, mouse||59, Multiple|
|Methionine- and choline-deficient diet (MCD)||NASH||Panlobular||Rat, mouse||116|
|MCD+high fat in OLEFT rats||NASH, insulin resistance||Panlobular||Rat||40|
|L-SACC1 mice+high fat||NASH||Panlobular||Mouse||38|
Interpretation of Results from Animal Models.
It is generally accepted that a promising drug will have to show efficacy in at least two mechanistically distinct fibrosis models to exclude “model-specific” artifacts. Additionally, the minimal quality requirements for satisfactory preclinical testing are (1) the use of sufficient numbers of animals to overcome individual heterogeneity in fibrosis progression (n = 8–15 per group depending on the model); (2) analysis of liver samples of sufficient size (5–10% of the liver) to eliminate considerable sampling variability; and (3) use of complementary quantitative and semiquantitative parameters to determine fibrosis, fibrogenesis, and fibrolysis by way of: (i) total collagen as measured by hydroxyproline content. (ii) morphometrical connective tissue assessment (Sirius Red staining); (iii) liver architecture as assessed by semiquantitative scoring; (iv) quantitative reverse-transcription polymerase chain reaction (RT-PCR) for transcripts related to fibrogenesis and fibrolysis; and (v) proteolytic activities. Specific attention needs to be paid to the genetic background of the mice, which influences their susceptibility to develop liver fibrosis. Therefore, the choice of a susceptible strain for fibrosis studies, the purity of the genetic background, and the use of wildtype littermates as controls are central for the interpretation of results.
Animal Models of Biliary Fibrosis.
The model of secondary biliary fibrosis due to bile duct ligation and scission (BDL) in rats is widely accepted, technically simple, and applicable also to mice. It results in a reproducible portal tract fibrosis over a short period of time (4–6 weeks in rats, 2–3 weeks in mice). However, it shows little resemblance to human fibrotic liver diseases (except for chronic biliary obstruction) and an altered metabolism of most drugs that are primarily secreted through the biliary tract or that undergo enterohepatic circulation. Therefore, mice that lack the hepatocyte phospholipid flippase Mdr2 (abcb4)−/−, the mouse homolog of the human Mdr3 gene) are increasingly used. They develop biliary fibrosis due to cholangiocyte proliferation and massive up-regulation of profibrogenic genes as early as 4 weeks postpartum.34 Fibrosis progression is spontaneous (i.e., does not require manipulation or toxin administration), progressive, and highly reproducible. Furthermore, it resembles human primary sclerosing cholangitis, while maintaining features common to other advanced chronic liver diseases including the development of liver cancer at 8–10 months of age.
When mice are fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 6–8 weeks, they develop pronounced aberrant ductular proliferations, similar to Mdr2−/− mice. The model was recently characterized in several mouse strains,35 and is especially attractive because it can be applied in genetically modified mice.
Hepatotoxin-Induced Animal Models of Liver Fibrosis.
Liver fibrosis induced by carbon tetrachloride (CCl4) results from repetitive (necrotic) hepatocyte death and continues to be popular because it allows the study of acute liver injury, advanced fibrosis, and fibrosis reversal (reviewed in Ref.3). Although mice generally show less toxin-induced fibrosis than rats, the different susceptibility of inbred strains to CCl4-induced liver fibrosis is well characterized.36 The model's major disadvantages are the severe hepatocyte necrosis and its dependence on massive oxidative stress that is not found to such an extent in human chronic liver diseases. A similar degree of fibrosis can be achieved by chronic injection of the hepatotoxin thioacetamide (TAA). Although it is essentially impossible to separate antiinflammatory or free radical scavenging activities of drugs from antifibrotic effects when these are given during toxin administration, the toxin models are very useful to test the efficacy of agents to speed up fibrosis reversal after toxin administration has been discontinued. Furthermore, the pattern of fibrosis achieved closely resembles the panlobular and parenchymal fibrosis that is found in most human chronic liver diseases.
Animal Models of NASH.
More recently, several models have been described that mimic important aspects of human NASH and fibrosis, in combination with features of the metabolic syndrome.37–39 The most efficient models exploit a genetic predisposition for the metabolic syndrome in combination with a high-fat diet. When a methionine choline-deficient diet was added as a third “hit,” obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats developed cirrhosis within 8 weeks.40
Transgenic and Gene Deletion In Vivo Models.
Genetic models have great value to confirm factors and mechanisms that drive fibrogenesis or fibrolysis in vivo, even more because the models have been refined with the use of tissue- and cell-specific, or conditional transgenes or deletions. An example are transgenic mice with constitutive overexpression of PDGF-C,41 or with conditional overexpression of TGF-β42 or PDGF-B.43 However, these models need further refinement because expression of the profibrogenic factors is typically driven by way of the albumin promoter in hepatocytes, whereas physiologically they are mainly produced by inflammatory cells and activated HSC.
Antifibrotic Drug Development
Impressive progress in our understanding of molecular mechanisms of fibrosis progression uncovered several promising molecular targets for antifibrotic treatments (Table 3) (for reviews, see Schuppan and Afdhal,1 Friedman,5 Bataller and Brenner,6 and Rockey44). However, transfer of these experimental treatments to clinical practice has been slow.
|TGFβ||HSC, cholangiocytes, inflammatory cells, endothelia||Soluble type II receptor, anti-TGFβ antibody, anti-TGFβ small molecule antagonists||46, 47|
|Integrin αvβ6||Activated epithelia||Small molecule antagonist, blocking AB||17, 26, 51|
|Endothelin-A receptor||Cholangiocytes, HSC||Small molecule antagonists||117|
|Cannabinoid receptors CB1, CB2||Cholangiocytes, HSC||Small molecule CB1 antagonistsCB2 agonists||118|
|Toll like receptor-4 (TLR-4)||Macrophages, HSC||Small molecule antagonists (also to downstream targets)||119|
|Angiotensin-1 receptor, angiotensin converting enzyme (AT1/ACE)||HSC||Numerous small molecule inhibitors||6|
|Caspases||Hepatocytes||Small-molecule inhibitors of caspase activation||120|
|Farnesoid receptor (FXR)||HSC||FXR agonists||121|
A major obstacle to antifibrotic drug development has been the usually slow progression of liver fibrosis in humans, coupled with a lack of sensitive and noninvasive means to assess fibrosis or fibrogenesis (see below). Furthermore, many patients are asymptomatic even when fibrosis is already advanced, or present with decompensated cirrhosis, which would require not only inhibition but also reversal of established fibrosis. Accordingly, antifibrotic drug development must consider the following two scenarios, balanced against the risk, cost, ease, and duration of treatment: (1) long-term: mainly inhibiting further progression, using a low-risk, relatively cheap oral agent; (2) short-term/interval: inducing reversal of (decompensated) disease or incipient cirrhosis, using a potentially high risk, parenteral, and possibly expensive agent.
Targeted approaches are directed to known molecular targets or pathways that are critically involved in fibrogenesis or fibrolysis, and that do not overlap significantly with unrelated pathways (which would incur unwanted side effects). Such agents can be function-blocking antibodies, or preferably small molecule inhibitors. A limitation of the antibody approach is its restriction to cell surface or extracellular targets and an often low bioavailability at the target site. However, they may allow rapid proof of principle testing in vivo. Small molecules are usually obtained by way of screening of existing compound libraries in a cellular readout system, followed by further chemical refinement. This process is effort- and time-consuming, but may yield orally available drugs. However, most of these developments never reach clinical phase II-III studies due to unwanted (off-site) side effects.
Bioinformatics is gaining importance in drug development and improvement, especially as in silico drug engineering and predictive activity modeling.45 This approach is based on 3-10 compounds with known activity toward a target. Affinities of these compounds have to be known, but an in-depth understanding of drug-target interactions is not required. From a virtual library of several million compounds, structure-affinity comparisons and in silico optimization will yield novel lead compounds that are synthesized and tested in a molecular or cellular readout assay for affinity, specificity, and other desired properties. Subsequently, these improved compounds will then be reentered into a novel cycle of in silico optimization and testing.
Several targeted approaches have been directed toward the profibrogenic TGFβ signaling pathway, e.g., using a soluble TGFβ receptor type II,46 TGFβ blocking antibodies, TGFβ antisense oligonucleotides, or molecules that interfere with downstream (smad-mediated) signal transduction. Two monoclonal antibodies against TGFβ1, Lerdelimumab and Meselimumab, are currently in clinical phase I-III trials in pulmonary fibrosis, systemic sclerosis, and postoperative scarring in glaucoma patients.47 There is an important limitation in targeting the TGFβ pathway systemically (this also applies to other physiologically relevant targets such as tissue inhibitor of metalloproteinases [TIMP-1] or certain matrix metalloproteinases [MMPs]), because apart from stimulating wound healing and fibrosis, TGFβ is also a central inhibitor of uncontrolled inflammation, and essential in inducing epithelial differentiation and in triggering apoptosis. This raises safety concerns for the general and long-term use of TGFβ inhibition, especially in patients with chronic (hepatic) inflammation. Another yet preclinical strategy is to block TIMP-1, a major effector of liver fibrosis, by use of a recombinant mutant protein derived from its high-affinity ligand MMP-9.48 An intriguing novel target is the integrin αvβ6, a cell surface receptor that is expressed on activated epithelia during embryogenesis, wound healing, and tumorigenesis.49 αvβ6 anchors these epithelia to a provisional ECM of tenascin and fibronectin17 and through tethering latent TGFβ1 promotes its proteolytic activation.50 In the liver, αvβ6 is exclusively expressed on activated cholangiocytes that are potent promoters of liver fibrogenesis.17, 26, 51 Its successful targeting by a blocking antibody51 or a small molecule inhibitor specifically blocked proliferation of profibrogenic cholangiocytes and concomitant TGFβ1 activation at the site of fibrogenesis.17, 26 Promising targeted approaches are summarized in Table 3.
Alternative and Complementary Medicine.
Alternative medicines for chronic liver disease, mostly herbal preparations from single or multiple plants, can be traced back 4000 years in ancient China and India and hold promise for the development of cheap and potentially low-risk “natural” drugs. Numerous studies, mainly in journals that are not easily accessible, report antifibrotic effects of herbal medicines. However, there is a slowly growing number of several better-defined active ingredients from herbal medicines that show antifibrotic properties, such as baicalein, curcumin, and silymarin52, 53 (Table 4). Although over-the-counter botanicals represent a multibillion market in the US alone, significant progress in this area is hampered by two major obstacles. First, the reliance of traditional Eastern medicine on empiricism and a holistic philosophy, where controlled studies are not considered necessary and where particular herbs are believed to be active only in certain combinations. This is in conflict with the Western approach, which relies on scientific evidence requiring adequately designed, placebo-controlled, randomized trials to prove the efficacy of structurally defined agents rather than of complex herbal mixtures.52 Second, the liberal use of poorly standardized crude extracts or complex mixtures of botanicals poses an increased risk of hepatotoxicity.54 As a result, only a few botanicals are currently in recognized clinical trials, including the flavonoids silymarin or silibinin from milk thistle for patients with chronic hepatitis C or nonalcoholic steatohepatitis55 (see http://clinicaltrials.gov/ for a complete list).
|Active Compound||Source||Mode of Action||References|
|Silibinin-1/-2 (Silymarin)||Milk thistle||Antioxidant, TGFβ-inhibition, hepatitis C viral suppression||52, 55, 122|
|Baicalein||TJ-9 (Sho-saiko-to)||Antioxidant, inhibition of HSC proliferation||123|
|Curcumin||Turmeric||Antioxidant, NF-kB and TNFα-inhibition, inhibition of HSC proliferation, induction of HSC apoptosis||124|
|Salvianolic acid, tanshinones||Salvia miltiorrhiza||Antioxidant, TGFβ-inhibition||126|
|Berberin||Coptis||Antioxidant, activation of superoxide dismutase||127|
|Trans-resveratrol||Red wine||Antioxidant, NF-κB inhibition||128|
Drug repositioning is the effort to develop treatments by finding novel applications for drugs that are already in clinical use for other indications.56 Examples for liver fibrosis are immunosuppressants such as rapamycin,57 antidiabetic glitazones,58 the antiparasitic drug halofuginone,59, 60 the tyrosine kinase-targeted anticancer drugs sunitinib,61 or imatinib mesylate (Gleevec),62 and blockers of the angiotensin system.6 Because the clinical safety and efficacy profile is already known for these agents, drug repositioning offers the chance of a “fast track” introduction of novel treatments into clinical practice. However, their antifibrotic efficacy in vivo may be limited, requiring either their combination with other drugs, or their targeted delivery to relevant liver cells.
Targeted Drug Delivery to Fibrogenic Liver Cells.
Effective targeting will increase local drug concentrations and diminish potential side effects. By nature, activated HSC are a major target for antifibrotic therapies, but drug targeting to HSC has been difficult because cell surface molecules up-regulated during fibrogenesis and inflammation, such as the mannose 6-phosphate receptor and certain integrins, are also found on other liver cells, such as hepatocytes, inflammatory, and endothelial cells.63 A few exceptions, such as the PDGFβ receptor or the collagen VI receptor, are mainly expressed on activated HSC and are virtually absent from normal liver.13, 64 Thus, the immunosuppressive and antiproliferative drug mycophenolate mofetil65 or apoptosis-inducing gliotoxin66 was coupled to mannose6-phosphate (insulin-like growth factor type II) receptor binding constructs that after intravenous injection mainly targeted HSC and sinusoidal endothelial cells. Coupling proapoptotic gliotoxin to a single-chain antibody against synaptophysin, which is expressed in activated HSC-induced apoptosis of activated HSC, and thus reduced fibrosis in the CCl4 model.67
Alternatively, vitamin A-modified liposomes increased delivery of small interfering RNA (siRNA) directed against the putative collagen-specific chaperone HSP47 to vitamin A-storing HSC, causing a pronounced antifibrotic effect in the CCl4 and BDL-induced liver fibrosis.68 Once effective targeted delivery of siRNA to activated HSC is confirmed, the versatility of this platform holds promise, because it permits the silencing of any known profibrogenic gene. Recently, targeting of adenovirus to the PDGFβ receptor using the receptor binding octapeptide (CSRNLIDC) cloned upstream of a single-chain antibody fragment directed against the adenoviral knob was employed to redirect adenoviral delivery of siRNA from hepatocytes to activated HSC.69
Noninvasive Means to Assess Liver Fibrosis and Fibrogenesis
The major obstacle to antifibrotic drug development has been the difficulty in defining accepted surrogate endpoints for clinical trials. A slowly evolving disease (many years to several decades for the development of cirrhosis, hepatic decompensation, or hepatocellular carcinoma development) or an elusive endpoint (liver biopsy) represents a significant hurdle for study design. Notably, mere inhibition of fibrosis progression (i.e., without the above-mentioned hard endpoints) is only slowly becoming accepted by regulatory authorities, such as the Food and Drug Administration (FDA).
To date, clinical studies on the progression or regression of liver fibrosis, with or without potential antifibrotic treatment, have relied on sequential liver biopsies and assessment of fibrosis progression using the conventional staging systems. Liver biopsy is invasive and risky,70 samples only 1/50,000 of the liver, and is prone to considerable sampling error. This is illustrated by a study that compared laparoscopic liver biopsies from the right and the left lobe in patients with hepatitis C. One-third of paired samples differed by at least one out of the four Metavir stages, and 14.5% showed advanced fibrosis in one and cirrhosis in the other lobe (stage F3 versus stage F4).71 Construction of smaller virtual biopsies from surgical liver biopsies confirmed that in hepatitis C even large biopsy cores (>25 mm) confer an inherent one-stage sampling variability of 25%.72 Even higher sampling variabilities are reported for NASH and biliary fibrosis.73, 74 Therefore, the following requirements must be fulfilled for clinical studies that use conventional staging of liver biopsy alone in order to prove a significant antifibrotic drug effect: (1) at least 200-500 well-selected patients with a single etiology of their liver disease; (2) patients with an intermediate stage of fibrosis, preferably Metavir F2, which serves as the best predictor for further fibrosis progression while still permitting a sensitive detection of drug-induced changes; and (3) a study duration of at least 2-5 years, with pre- and poststudy biopsies. Thus, based on staging alone, even exploratory phase I and II studies are hampered by the need for a significant sample size and by a high risk of failure. However, even with currently available techniques, patient numbers and study duration may be reduced significantly, pending prospective validation against hard endpoints. Thus, conventional biopsy readouts can be improved, such as by semiquantification of activated myofibroblasts by way of staining for α-SMA or TGFβ,75 or by quantitative PCR quantification of transcripts that are related to fibrogenesis or fibrolysis.76 When assessing progression with or without therapy, a cohort of, for example, HCV-infected patients with intermediate fibrosis (Metavir stage 2) would be preferable, because this would best allow detection of modest changes in fibrous tissue in a population with a high likelihood of progression. Antifibrotic effects will be more difficult to detect in patients with precirrhotic or cirrhotic disease, because collagen content increases exponentially with each stage, resulting in a roughly 4 to 5-fold increase in liver collagen in stage 4 (cirrhosis) over stage 2.72 On the other hand, patients with cirrhosis obviously need an antifibrotic therapy most urgently. In these patients, a functional parameter that is correlated with the extent of remodeling and fibrosis, such as hepatic portal vein pressure gradient (HVPG) is plausible.77 In fact, a recent study confirmed HVPG as the best predictor of mortality in cirrhotic patients with compensated Child's A cirrhosis.78
Yet the shortcomings of traditional study design and technologies highlight the need for the identification of new biomarkers or imaging techniques that allow an exact assessment of the degree of fibrosis and, more important, of the dynamic processes of fibrogenesis or fibrolysis, in order to predict fibrosis progression or to monitor the effect of antifibrotic therapies.
Serum Fibrosis Markers.
The recent literature is replete with studies that employ serological fibrosis markers and their combinations to cross-sectionally stage liver fibrosis (reviewed in 1, 79-81). These have mainly been validated in chronic hepatitis C and can show diagnostic accuracies around 80% for the differentiation between no or mild (Metavir F0/1) and moderate to severe (Metavir F2-4) fibrosis. Their inability to differentiate intermediate fibrosis stages, e.g., stage 1 from 2, precludes their use as surrogates in clinical studies with potential antifibrotics, a setting in which fibrosis progression is slow. Furthermore, these markers are only used for a dichotomous differentiation of patients into two crude (no/mild vs. moderate/severe) fibrosis categories, whereas fibrosis progression is nonlinear.82, 83 Furthermore, even when using the dichotomous classification there is a high indeterminate rate with an inability to stage fibrosis in 30%–70% of patients with intermediate fibrosis stages. To complicate matters, the amount of hepatic fibrous tissue increases exponentially with every fibrosis stage,72 whereas these surrogates are continuous variables. Finally, for their validation these markers and algorithms have to be compared to liver biopsy, which, as mentioned above, is far from an ideal “gold standard” due to inherent sampling variability. Given the drawbacks of liver biopsy, a recent study calculated the chance to identify a given marker panel to correctly stage fibrosis according to the above-mentioned criteria.84 By assuming the realistic scenario of 90% sensitivity and 80% specificity of liver biopsy to correctly identify significant fibrosis F ≥ 2, even a perfect serum test (accuracy 99%) would result in a test accuracy below 80%, well in the range of current marker panels. The fibrosis markers and their combinations have almost exclusively been validated cross-sectionally, i.e., in comparison to fibrosis stage, whereas those markers that are related to matrix metabolism may rather reflect dynamic alterations of connective tissue turnover, i.e., fibrogenesis or fibrolysis. Thus, several studies suggest that certain marker combinations, such as Fibrotest, the enhanced liver fibrosis panel of ECM markers (ELF), or other selected ECM markers may have more value in predicting progression rather than fibrosis stage.85–90 However, their validation as dynamic markers is difficult and will require more long-term studies. Such studies should also be complemented by determination of hepatic fibrogenesis by quantitative PCR for transcripts that are related to fibrogenesis or fibrolysis in matched liver biopsies.
Attempts to Discover Better Serum Markers Using Proteomics and Transcriptomics.
Molecular alterations associated with liver fibrogenesis or fibrolysis often involve secreted proteins, making it likely that specific biomarkers for different phases of the disease do exist in serum. Proteomic technologies based on protein fractionation and mass spectrometry (MS), with the aid of bioinformatics, provide the means to compare protein profiles in normal and pathological serum. Serum is depleted of up to 20 of the most abundant proteins, resulting in <2% of the original protein content but >99% of original protein variety. Depleted sera are then either separated by 2D gel electrophoresis (DIGE) that allows comparative analysis of three different samples in a single gel, or by tryptic digests of serum proteome fractions that have been labeled with so-called isobaric tags (iTRAQ technology), permitting parallel analysis of up to eight serum samples.91–93 Identification of the in gel trypsin digested proteins (DIGE) or of the tryptic iTRAQ peptides is done by MS. The power of these technologies lies in a quantitative comparison of an identified peptide or protein in three (DIGE) or eight (iTRAQ) serum samples. However, their sensitivity to detect proteins at the lower end of serum concentrations, i.e., in the picomolar range, such as cytokines or ECM peptides or proteins, is still insufficient. Furthermore, differential glycosylation generates complex patterns for a single protein in DIGE, which is based on charge separation in the first dimension.
Nonetheless, projects that employ proteomics are on the way, not only in serum or plasma samples that can be correlated to readouts of matched biopsies, but also in rodent models of fibrosis progression or reversal. The latter approach is promising, because mechanisms and molecules in fibrogenesis and fibrolysis in rodents are highly similar to those in humans, and representative liver tissue can be sampled for correlation to the extent of hepatic fibrogenesis or fibrolysis. This allows the transfer of the newly discovered biomarkers rapidly to the human system.
Novel serum markers may also be found by analysis of the liver transcriptome, an approach which has been attempted mainly in biopsies from patients with hepatitis C.94, 95 This technology is far simpler than serum proteomics and may detect low abundancy gene expression. However, most of the regulated gene products will not be shed into the circulation and are unspecific or unrelated to the fibrogenic process, requiring a high effort to find and validate a single novel serum marker.
Imaging of Liver Fibrosis.
Imaging of liver fibrosis, and in particular hepatic fibrogenesis, with conventional methodology has been elusive. Ultrasonography, computerized tomography, magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computerized tomography (SPECT) are not able to detect fibrosis and are not even sensitive enough to diagnose cirrhosis in many patients.1 Diffusion-weighted MRI may offer some differentiation, but will likely not be able to exactly stage fibrosis.96 MR texture analysis and double-contrast MRI using supraparamagnetic iron and gadolinium as contrast agents may yield a better fibrosis assessment but are in their infancy.97, 98 Recently, a functional MRI method was proposed that is based on measuring hemodynamic response in the liver to hypercapnia and hyperoxia. Increase in liver perfusion was attenuated according to degree of fibrosis in rats.99 This approach, if validated in patients with liver disease, may offer an additional noninvasive diagnostic tool for evaluation and follow-up of liver diseases.
Ultrasound Elastography (Fibroscan).
This is a novel, noninvasive bedside method to assess liver fibrosis by measuring hepatic stiffness. An intercostally placed probe transmits low amplitude shear waves through the liver, and their velocity is picked up by an integrated ultrasound device (pulse-echo ultrasound). Shear wave velocity is inversely correlated with liver stiffness. The method is painless and without risk, takes only 5 minutes, and can be performed in a fairly standardized way after only a short training period. Furthermore, it measures a novel, i.e., mechanical quality of the liver related to fibrosis and, when compared to biopsy, samples a 100-fold larger volume (4 × 1 cm, ≈1/500 of the liver). More than 100 clinical studies correlating ultrasound elastography with liver biopsy have appeared since its first description in 2003,100 including meta-analyses.101–103 These show a good correlation of stiffness values with the histological stage of fibrosis. Area under receiver operating curves (that reflect the diagnostic precision in select populations) yielded excellent accuracy for differentiating cirrhosis from noncirrhosis (AUROC between 0.90 and 0.99), especially for patients with chronic hepatitis C. However, with the practical but crude histological Metavir staging system (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 sampling variability. In addition, severe inflammation and mechanical cholestasis can significantly increase hepatic stiffness, thus confounding the fibrosis readout.104–106 Nonetheless, ultrasound elastography should prove useful for stratification of patients for inclusion into treatment studies.
MR elastography is based on similar principles as ultrasound elastography, but studies are few and most have not included low numbers of patients. A larger generator transmits the shear waves which are recorded with a 1.5 T MR scanner.107 Advantages are examination of the whole liver, an even lower observer dependency, and applicability in patients with severe obesity or narrow intercostal spaces (which can preclude ultrasound elastography). A disadvantage is the more restricted availability. Importantly, acquisition times can be shortened 10-fold (20 to 2 minutes) by use of echo-planar versus spin-echo sequences, without compromise of the stiffness readout.107 A recent study in 133 patients with various liver diseases revealed stunning AUROC values between 0.985 and 0.998 for the diagnosis of fibrosis stages F ≥ 2, F ≥ 3, or F ≥ 4, and superiority of MR elastography over ultrasound elastography or a combination of the serological AST over platelet ratio (APRI) combined with ultrasound elastography.108 Hepatic inflammation and mechanical cholestasis are expected to be confounders as with ultrasound elastography, but studies are lacking.
Molecular Imaging of Liver Fibrosis and Fibrogenesis.
Molecular (targeted) imaging technology is based on a high-affinity ligand for a cell surface molecule that has 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 is particularly relevant for imaging of fibrosis or fibrogenesis. In addition, the imaging construct should be nontoxic and have a desirable plasma half-life of 10–30 minutes, to be eliminated predominantly through the kidneys. Finally, unspecific background uptake should be low to yield a good signal-to-noise ratio. Attractive target molecules are the abundant fibrillar collagens (for fibrosis measurement) and cell surface markers of fibrogenesis such as integrin αvβ6 on cholangiocytes and the PDGFβR on activated HSC for quantification of fibrogenesis (Fig. 3). The availability of quantitative fibrogenesis imaging over the whole liver could enable the testing of antifibrotic agents in small numbers of patients and over short periods of time. Early data on the feasibility of this approach are emerging.110
- Top of page
- Promising Targets for Antifibrotic Therapies
- Preclinical Models to Test Potential Antifibrotics
The stage appears to be set for a highly predictive and effective preclinical selection and testing of antifibrotic agents. Their clinical validation in phase I and II studies will be jump-started once the desired noninvasive methods for their rapid and reliable testing in small numbers of patients have been developed.
- Top of page
- Promising Targets for Antifibrotic Therapies
- Preclinical Models to Test Potential Antifibrotics
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