Curr Treat Options Gastro DOI 10.1007/s11938-015-0053-z

Liver (J Bajaj, Section Editor)

New Medical Treatment Strategies for Nonalcoholic Steatohepatitis Michael Fuchs, M.D., Ph.D., F.A.A.S.L.D., A.G.A.F., F.E.B.G. Address Hunter Holmes McGuire Department of Veterans Affairs Medical Center, Gastrointestinal & Hepatology Service (111-N), 1201 Broad Rock Boulevard, Richmond, VA 23249, USA Email: [email protected] 2 Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University Medical Center, Richmond, VA 23298, USA

* Springer Science+Business Media, LLC 2015

This article is part of the Topical Collection on Liver Keywords NASH I Nuclear receptors I Microbiome I Liver fibrosis I Inflammation I Hormones

Opinion statement Nonalcoholic steatohepatitis (NASH) is a severe form of fatty liver disease unrelated to chronic alcohol consumption. As nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions and is becoming the most common cause for chronic liver disease, NASH likely will replace chronic hepatitis C as leading indication for liver cirrhosis and liver transplantation in this decade. Despite this alarming trend, effective treatment is lacking and continues to rely on dietary interventions and physical exercise, known to be of limited effect. Hence, there is an urgent need for safe pharmacologic therapy that successfully reverses or prevents progression of liver injury and fibrosis in patients with NASH. Employing emerging concepts of disease development and progression that involve in parallel ongoing events originating from the liver, adipose tissue, and intestine will ultimately promote the development of effective agents for targeted therapies for NASH. Novel agents must be safe, optimally dosed, and finally pass clinical trials providing proof of concept and efficacy. In the case of NASH, trial design is not necessarily straightforward as surrogate end points to predict clinical benefits are not yet established. The level of unmet needs for NASH goes however even beyond therapeutic agents and also includes patient and physician awareness. This article focuses on identifying potential pathophysiology-guided targets for medical therapy of NASH.

Introduction Until 1980, storage of fat in the liver was considered essentially benign and the term nonalcoholic fatty liver

disease (NAFLD) did not even exist. NAFLD is defined by the pathological accumulation of fat (95 % of the

Liver (J Bajaj, Section Editor) hepatocytes) in the absence of significant alcohol consumption and by excluding secondary causes of hepatic steatosis [1]. Two principal phenotypes are recognized: (i) simple (Bbland^) macrovesicular steatosis or nonalcoholic fatty liver (NAFL) and (ii) nonalcoholic steatohepatitis (NASH) characterized by inflammation and hepatocyte ballooning injury associated with varying degrees of fibrosis. Natural history data suggest that NASH is a prerequisite to develop liver cirrhosis contributing to a large proportion of cryptogenic cirrhosis. Emerging evidence supports the concept that progression of NASH to end-stage liver disease results from parallel events originating from the liver, the adipose tissue, and the gut. By most estimates, about 40 % of the US general population has NAFLD and about 10 % of them are believed to have NASH [2–4]. A similar prevalence is believed to exist in the rest of the Western world [5]. With the epidemic of obesity and diabetes as well as the capability to cure hepatitis C, NASH is positioned

to become the leading cause of chronic liver disease. It is without doubt that NAFLD increases all-cause mortality [6] with liver-related mortality representing the third most common cause of death in patients with NAFLD [7]. NASH contributes substantially to a rapidly growing indication for liver transplantation particularly in patients with liver cancer [8]. Not entirely surprising, cardiovascular disease has been identified as the leading cause of death [9]. Keeping this in mind, we likely underestimate the relevance of this disease as a cause of end-stage liver disease, clearly highlighting the need for effective therapy for NASH. As our therapeutic options are currently not only very limited but also not very effective, establishing treatment targets and discovering compounds for targeted therapy resulted in a recent surge of interest of pharmaceutical companies to develop drugs for NASH. This review will highlight novel treatment targets for which compounds may be developed or have already entered the clinical trials arena.

Treatment Goals of therapy and treatment strategy Developing an effective NASH treatment from the original idea, selection of the best target, identification, and optimization of a candidate molecule for clinical development resulting in the launch of a finished product is a costly and often long and complex pathway. Drug development programs should consider early on clinically relevant as well as achievable end points [10•]. For NASH drug development, one should focus first on reversing steatohepatitis either defined by improvement of the NAFLD activity score or disappearance of necroinflammatory lesions associated with progression of the disease. This end point is early in the natural history of the disease and can be achieved in trials with reasonable sample sizes and duration. Antifibrotic therapy aims either at preventing development of or reversing liver cirrhosis. Documentation of clinical benefit for these so-called hard end points requires large outcome trials and is harder to achieve. NASH development and disease progression to cirrhosis involve not only a complex interplay between several signaling pathways, but also, the contribution of each pathway to disease severity and progression is unknown and may vary among patients. Therefore, selection of a single target for therapy likely needs to be at a point where pathways converge. Alternatively, and more likely, targeting more than a single pathway will be needed for effective therapy. It may be that therapies addressing inflammation as well as fibrosis will turn out to be the solution needed to have a significant impact in this field.

New approaches for therapy NASH is a systemic disease with complex metabolic alterations affecting the liver and other organs such as the adipose tissue and intestine. Disease

Novel Therapies for NASH progression is a function of liver cell injury and the possibility of tissue restitution versus development of fibrosis associated with progressive architectural destruction of the liver. Key contributors to disease progression within or outside the liver have been identified and may represent targets for treatment that are discussed below. In the following, evolving treatments for NASH and approaches that target key pathophysiological mechanisms of this common metabolic liver disease will be discussed (Table 1).

A. Treatments directed at mechanisms of hepatocellular lipid accumulation and insulin resistance There is an intricate relationship between steatosis and insulin resistance, a hallmark of nonalcoholic fatty liver disease. Clearly, insulin resistance in general can cause hepatic steatosis, whereas hepatic steatosis may promote insulin resistance under select circumstances [11]; alternatively, both steatosis and insulin resistance just reflect the presence of obesity [12], a fact that could be of critical importance when designing targeted therapy for NASH. It is very obvious that hepatocellular accumulation of triglycerides occurs very early in the development of NASH. Although a prerequisite for NASH development, steatosis per se does not necessarily result in liver damage. In fact, by means of esterification of free fatty acids, either from diet or released from adipose tissue, synthesis and storage of triglycerides will prevent lipotoxicity from otherwise accumulating free fatty acids [13]. It is believed that lipotoxicity occurs once the capability to upregulate triglyceride synthesis cannot keep up with the excessive flow of free fatty acids. Under these circumstances, free fatty acids may interfere with insulin signaling [14] and oxidative stress overwhelms compensatory defense mechanisms which results in hepatocellular damage. Inhibiting triglyceride synthesis under these conditions may actually be harmful [15]. For that exact reason, it is plausible that solely targeting steatosis may be of limited benefit. It also raises the question if steatosis is the innocent bystander to concomitantly developing steatohepatitis. If this would be the case, then simply reducing triglyceride storage may not represent an attractive target for therapy. Further clarification may come from exploring ISIS-DGAT2Rx, an antisense drug specifically targeting the production of triglycerides by blocking diacylglycerol acyltransferase 2. Aramchol, a fatty acid-bile acid conjugate (3β-arachidyl-amido,7α-12αdihydroxy,5β-cholan-24-oic acid) inhibiting stearoyl coenzyme A desaturase 1 (SCD1), is capable to facilitate removal of liver fat [16] in animals. This resulted in a short-term randomized, double-blind, phase 2 study in patients with NAFLD. Results of this study demonstrated improvement in liver fat content as measured by magnetic resonance spectroscopy [17]. Based on these findings, future long-term clinical trials need to be initiated in a larger patient population that include histological assessment of treatment response. Interestingly, the FDA granted fast-track status for aramchol for the treatment of NASH; fast-track designation is granted to those compounds that are considered to be capable of fulfilling an unmet medical need and usually will expedite the review process in the future. Thiazolidinediones improve insulin sensitivity and have been investigated in patients with NAFLD, the largest trial using pioglitazone in nondiabetic patients with biopsy-proven NASH [18•]. Although histological improvement has been observed, weight gain and concerns about long-term safety [19–21]

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Table 1. Selected compounds in development for NASH. Summary of the developmental stages of selected compounds evaluated in patients with NASH. For abbreviations, please refer to the text Compound

Target

DGAT2Rx AM6545 JD5037 LJPC-1010 PXS-4728A LY2405319 GKT137831 Simtuzumab Cenicriviroc Emricasan Liraglutide Sitagliptin Exenatide Remogliflozin etabonate Aramchol Px-102 MBX-8025 GR-MD-02 GFT505

DGAT2 CB1 receptor CB1 receptor Galectin-3 LOXL2 FGF21 NOX1/4 LOXL2 CCR2/CCR5 Caspase GLP-1 DPP-4 GLP-1 SGLT2 SCD1 FXR PPARδ Galectin-3 PPARα/δ

Application

Development stage

Company

Oral Oral s.c. Oral i.v. Oral Oral s.c. Oral s.c. Oral Oral Oral Oral i.v. oral

preclinical preclinical preclinical Phase 1 Phase 1 Phase 1 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2

ISIS Pharmaceuticals MAKScientific LLC Jenrin Discovery Inc. La Jolla Pharmaceuticals Pharmaxis Eli Lilly Genkyotex Gilead Sciences Tobira Conatus Novo Nordisk Merck Astrazeneca Islet Sciences Inc. Galmed Phenex Pharmaceuticals Cymabay Therapeutics Galectin Therapeutics Genfit

require careful weighting of risks and benefits with careful monitoring of patients once on treatment. Therefore, there is a clear need for better drugs with the capability to improve insulin sensitivity. Inhibiting the renal sodium glucose transporter 2 (SGLT2) blocks glucose reabsorption and facilitates glucosuria thereby improving hyperglycemia [22]. SGLT2 inhibitors represent an important new class of compounds for metabolic diseases and are expected to be the only oral diabetes therapies to provide safe and robust insulinindependent HbA1c lowering with significant weight loss. Although there are numerous selective SGLT2 inhibitors currently under development for the treatment of diabetes mellitus, Remogliflozin etabonate appears to be unique. This drug not only displays insulin-sensitizing capacity but also has inherent antioxidant activity independent of its ability to inhibit SGLT2. This clearly differentiates Remogliflozin etabonate from other drugs in its class. It also has a significantly shorter half-life enabling a novel, once-daily biphasic-release formulation expected to yield optimal efficacy and tolerability profiles [23]. Therefore, Remogliflozin etabonate appears to be uniquely positioned as an effective therapy for patients with diabetes mellitus and NASH, but this needs to be confirmed in carefully designed clinical trials.

B. Liver-targeted treatments directed at mechanisms of hepatocellular injury Without question, NASH is an inflammatory disease with sustained inflammation being critical for fibrosis development and progression. Signals that result in hepatocellular injury may originate in the adipose tissue or the

Novel Therapies for NASH intestine and augment local inflammatory mechanisms within the liver involving Kupffer cells, monocytes, and lymphocytes. Whereas it is obvious that multiple signaling pathways are activated simultaneously, it will be quite a challenge to identify those pathways that are most relevant to disease progression and therefore best suited for targeted therapy. Increased expression of chemokines and their receptors, particularly C-C chemokine receptor 2 and its ligand CCL2, is a major regulator of macrophage recruitment to the liver during hepatic inflammation [24]. CCL2 or monocyte chemoattractant protein 1 is synthesized by a variety of cell types representing a central and redundantly activated pathway upregulated in NASH [25]. Other chemokines such as CCL5 may also play a role in NASH [26]. It is thus likely that a CCR2/CCR5 dual antagonist such as cenicriviroc may display antiinflammatory and potentially also antifibrotic effects [27]. This is currently being explored in a phase 2b proof-of-concept study enrolling more than 200 patients with biopsy-proven NASH. In this study, the primary end point after 1 year of therapy is improvement of steatohepatitis without worsening of fibrosis whereas the end point after 2 years is resolution of NASH also without worsening of fibrosis. Results of these trials should be available in 2016, and if positive, cenicriviroc would be the first specific anti-inflammatory intervention in NASH. Of interest is also the recent observation that the expression of another chemokine, CXCL16, is increased in NAFLD [28]. Experimental data support the concept that inhibition of the CXCR6-dependent accumulation of natural killer T cells in the liver may represent yet another pathway that could be targeted for treatment of NASH [28, 29]. An imbalance in the production and clearance of reactive oxygen species (ROS) causes oxidative stress, and, when chronic in diseases such as NASH, this is an important factor triggering fibrogenesis in the liver [30, 31]. Mitochondria are one of multiple sources that can produce ROS in the liver and in NASH particularly [32–34]. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multicomponent enzyme complex and the main source that generates ROS. Therefore, therapeutic benefit may be achieved by blocking NOX enzyme activity to suppress ROS production. Indeed, treating rodents with GKT137831 a first-in-class NOX1/4 inhibitor currently explored for the treatment of diabetic nephropathy, was able to inhibit hepatocyte apoptosis and attenuate liver fibrosis in a preventive and therapeutic way [35, 36]. Importantly, this inhibitor is designed in a way that it does not affect normal physiological processes such as host defense conferred by other members of the NOX family. Based on these encouraging results, a phase I study in patients with NASH is in preparation. Hepatocyte apoptosis or programmed cell death is a process required to maintain tissue health by counterbalancing cell proliferation and eliminating damaged cells. The presence of ALT in serum of healthy subjects supports the concept that the hepatocyte is constitutively undergoing proapoptotic stress. Although different apoptotic pathways are involved in NASH [37], they converge on activation of caspases, a group of cysteinyl aspartate-specific proteases, which play a central role in the execution of apoptosis and represent an attractive target for drug development. Because activation of caspases requires caspase activity, it is possible to design pan-caspase inhibitors with broad inhibition of all caspases. In NASH, the magnitude of apoptosis clearly correlates with the fibrosis stage, but the relative contribution of proapoptotic versus

Liver (J Bajaj, Section Editor) proinflammatory caspases is unknown [38, 39]. Based on this concept, decreasing hepatocyte apoptosis minimizes the secondary inflammatory response and subsequent liver fibrosis. Indeed, employing pan-caspase inhibitors VX166 and IDN-6556 (emricasan) in rodent models of NASH demonstrated improvement in liver fibrosis [40, 41•]. In patients with NASH, a 4-week therapy with the pan-caspase inhibitor GS-9450 decreased cytokeratin-18 fragments, a serum marker of hepatocyte apoptosis [42]. Although no safety concerns were raised for this short treatment duration, treating patients with chronic hepatitis C for 6 months with this inhibitor caused liver enzyme abnormalities that ended further development of this inhibitor. Emricasan (IDN-6556) has been successfully studied in patients with hepatitis C, and no relevant side effects were noticed [43] paving the way for a phase 2 study in patients with NASH, results of which are expected to be available in 2015. Although increasing cell viability by blocking apoptosis may raise concerns about potential malignant transformation, this appears unlikely. It is rather persistent apoptosis promoting inflammation with associated cellular proliferation that increases the risk of cancer, but careful long-term monitoring appears to be justified.

C. Treatments directed at mechanisms of liver fibrogenesis Employing in vitro as well as animal studies, great progress has been made in understanding the cellular basis of liver fibrogenesis. Yet, this has not translated into antifibrotic therapy in humans. While there are many reasons for the ineffectiveness of compounds explored so far, it is evident that, if possible at all, treating the underlying liver disease is the best antifibrotic therapy. As this appears to be more challenging in disorders with complex networks involved in disease progression such as NASH, the availability of antifibrotic therapy remains an unmet need. Galectin-3 is a multifunctional protein capable to bind to terminal galactose residues in glycoproteins and involved in multiple cellular processes such as inflammation, apoptosis, angiogenesis, and migration [44]. It is highly expressed in macrophages and involved in liver fibrosis as well as fibrogenesis in other organs. GR-MD-02 represents a complex carbohydrate drug containing terminal galactose residues that binds galectin-3 to inhibit its function. Encouraging studies in rodents have demonstrated that inhibition of galectin-3 was capable to decrease hepatic inflammation and fibrosis; moreover, reversal of liver cirrhosis with improvement of portal hypertension could be achieved as well. This finding illustrates that changing the liver architecture has a physiological effect on liver blood flow or resistance [45•, 46]. The mechanisms by which GR-MD-02 administration affects fibrosis are still unclear, but it is possible that the compound alters the macrophage population with a shift towards more reparative macrophages capable to degrade collagen. Based on these encouraging results in rodents, a phase I trial in patients with NASH and advanced fibrosis was initiated. Results demonstrated that GR-MD-02 given intravenously at doses up to 8 mg/kg was well tolerated and safe. After application of four doses, liver stiffness decreased, and this was in line with a concomitant improvement of a validated composite fibrosis score [47]. Based on these results, a phase 2 trial is planned for 2015. As GR-MD-02 may represent a potential breakthrough in the treatment of liver fibrosis, the company was granted BFast Track^ for its drug by the Food and Drug

Novel Therapies for NASH Administration. LJPC-1010 is another galectin-3 inhibitor with encouraging results in a NASH mouse model that showed significant improvement in steatosis, lobular inflammation, and hepatocyte ballooning. In contrast to GRMD-02, LJPC-1010 can be administered orally. For more than three decades, lysyl oxidase activity has been linked to liver fibrosis [48]. Lysyl oxidase-like 2 (LOXL2) belongs to the lysyl oxidase protein family responsible for cross-linking of collagen and elastin considered as final step in the fibrogenic pathway causing tissue stiffness and further enhancing transforming growth factor β tissue driven remodeling [49]. Simtuzumab (GS6624), a monoclonal antibody highly selective for LOXL2, is currently studied in patients with advanced liver fibrosis and cirrhosis caused by NASH. If this study is positive, PXS-4728A, a small molecule LOXL2 inhibitor with good oral bioavailability and once-daily dosing under development by Pharmaxis would be preferable compared to the infusion therapy required for simtuzumab. Endocannabinoid signaling in liver disease has gained attention over the last decade with the characterization of two G protein-coupled receptors CB1 and CB2. While CB1 receptors promote liver fibrogenesis and apoptosis in addition to increasing lipogenesis, CB2 receptors in striking contrast exert hepatoprotective effects due to anti-inflammatory and antifibrogenic properties [50, 51]. These opposing effects may suggest that in chronic liver disease, the fibrogenic CB1 signaling prevails on the antifibrogenic action of CB2. An important role of the CB2 receptor in NASH is supported by the presence of a functional receptor variant Q63R in obese children with NASH and particularly severe liver inflammation [52]. Therefore, inhibiting signaling through the CB1 receptor or activating the CB2 receptor pathway appears to be an attractive therapeutic strategy for the treatment of NASH. While the first-generation CB1 receptor agonist rimonabant showed encouraging results in NASH, its capability to pass the blood-brain barrier allowing it to act on central CB1 receptors caused considerable neuropsychiatric side effects [53] resulting in the withdrawal of rimonabant in 2008. Preclinical results obtained with the peripherally selective neutral CB1 receptor antagonist AM6545 and the inverse CB1 agonist JD5037 show promising results supporting to proceed with phase 1 studies.

D. Targeting nuclear receptor-mediated mechanisms participating in the development of NASH Nuclear receptors play a pivotal role in lipid metabolism, energy homeostasis, and inflammation and appear to be attractive therapeutic targets for the management of NASH and the insulin resistance that precipitates NAFLD. Indeed, combining several hepatoprotective properties in a single agent appears the most promising evolving approach in treating NASH. The challenge is to define both the optimal target(s) and the correct balance in receptor selectivity and binding affinity to ensure efficacy while minimizing untoward side effects. Bile acids have long been known to be involved in the regulation of lipid and carbohydrate metabolism, but they also display anti-inflammatory and antifibrotic activities [54]. The metabolic activity of bile acids appears to be mediated in large by activation of the farnesoid X receptor (FXR), a nuclear hormone receptor expressed at highest levels in the liver and intestine [55]; in addition, FXR controls the expression of genes with antimicrobial activities [56]. Developing FXR agonists for the treatment of NASH appears logic, and it should represent an elegant and effective way to readjust dysregulated nuclear receptor-

Liver (J Bajaj, Section Editor) mediated metabolic pathways. To optimize separation of desired therapeutic effects from undesirable side effects, it may be necessary to design organ- or gene-specific FXR agonists. Obeticholic acid, a 6α-ethyl derivative of chenodeoxycholic acid, is a first-in-class selective FXR agonist that has been studied in a large number of patients with biopsy-proven NASH [57•]. In a randomized, placebo-controlled study patients with biopsy-proven NASH received obeticholic acid for 72 weeks. The key finding of this trial is improvement in all histological features of NASH, and surprisingly in liver fibrosis as well. Resolution of steatohepatitis occurred almost twice as often as in the placebo group. Of potential concern, however, is the observation that treatment with obeticholic acid worsened the atherogenic profile and put subjects at increased risk for cardiovascular events. Clearly, this requires further exploration. Px-102 is a fully synthetic FXR agonist with a differentiated profile relative to obeticholic acid, and a phase 2 trial is currently underway in patients with NASH. As the nuclear receptor FXR is expressed not only in the liver, select activation of FXR in other organs such as the intestine may have the potential to maximize beneficial metabolic changes and minimize systemic side effects of systemic FXR activation. A first step into this direction is the development of fexaramine which is poorly absorbed from the intestine and selectively activating the FXR receptor in the intestine. Studies in rodents demonstrated that fexaramine protected against diet-induced weight gain lowered inflammatory cytokine levels and enhanced glucose tolerance as well as fatty acid oxidation [58•]. If this approach translates into human beings, then it could replace vertical sleeve gastrectomy, an effective surgical treatment option for NASH for which intestinal FXR has been identified as molecular target [59]. Peroxisome proliferator-activated receptors (PPARs) are a family of ligandactivated nuclear receptors that regulate lipid and glucose metabolism and in addition display anti-inflammatory as well as antifibrotic properties. Fibrates, known ligands for PPARα, are known for their lipid-lowering activities but have shown only limited efficacy in treating fatty liver disease [60]. Pioglitazone, a thiazolidinedione also referred to as glitazone, is a PPARψ agonist that has shown to exert beneficial effects in patients with biopsy-proven NASH, particularly in a subset of patients with weight loss [61]. However, long-term use of pioglitazone is problematic due to safety concerns [19–21]. There is a possibility that selective PPAR modulators may overcome some of these limitations, but progress has been rather slow. PPARα activation has the potential to enhance fatty acid oxidation and lipid catabolism and improve insulin resistance as well as inflammation [62]. Of potential interest for further development is MBX-8025, a selective PPARδ agonist which improves insulin resistance and liver function [63]. The most promising compound however is GFT-505, a PPARα/δ dual agonist with a quite unique pharmacokinetic profile of extensive enterohepatic cycling and accumulation in the liver. GFT-505 has shown efficacy towards improvement of insulin sensitivity, dyslipidemia, liver function, and fibrosis [64, 65, 66•]. GFT-505 is currently tested in a phase 2b randomized controlled study in patients with NASH; final results of which are eagerly awaited in spring 2015. Compared with obeticholic acid, its direct competitor in the field, GFT-505 may have a more favorable adverse effects profile with no

Novel Therapies for NASH pruritus and a more cardioprotective lipid profile. In 2014, the Food and Drug Administration granted fast-track designation.

E. Treatments targeting hepatic autophagy Autophagy or self-digestion, a process that targets cell constituents to lysosomes for degradation, is linked to insulin resistance, hepatic injury, and fibrogenesis [67]. Currently, there is evidence supporting a lipolytic as well as lipogenic effect of autophagy, and it very well may be that the metabolic state of a given tissue determines whether activation of autophagy results in lipolysis or lipogenesis [68]. At early stages of NAFLD, induction of autophagy may explain how caffeine ameliorates progression from steatosis to NASH [69]. Continuous altered autophagic flux however later could contribute to liver injury by providing free fatty acids from breaking down of triglycerides in lipid droplets within hepatocytes. A physiological defect in autophagy directly promoting NASH development has not been demonstrated yet. However, a recent study supports the concept that autophagy is increased in NASH [70]. A better understanding of the molecular mechanisms of autophagy and its implication in specific liver cells during the evolution of NASH may help identify new potential therapeutic targets [71, 72]. In this context, the recent discovered link between autophagy and nuclear receptors FXR and PPARα is of importance [73, 74]. It is possible that activation of these two transcription factors, currently being explored in clinical trials as therapeutic targets for NASH, antagonistically regulates how the availability of nutrients modifies the autophagic response in the liver. Clearly, much more basic work needs to be done before tissue-specific or systemic modulators of hepatic autophagy will be explored in clinical trials.

F. Correcting altered miRNA expression microRNAs (miRNAs) are small evolutionary conserved endogenous noncoding RNAs. Their primary function is the post-transcriptional regulation of target gene expression, and they have emerged as regulators of many physiological processes including lipid and glucose metabolism. It is thus not surprising that the hepatic mRNA expression in patients with NASH was altered and different from those patients with NAFLD [75]. Although it remains unknown if the miRNA dysregulation in NASH is cause or consequence of the liver disease, correcting miRNA levels could have immense therapeutic potential. A number of questions and challenges including specificity, delivery mode, and long-term consequences and others remain to be addressed first before this treatment approach may enter the clinical trial arena. Targeting specific miRNAs may also ameliorate NASH; however, the complexity of gene networks that a single miRNA may control and the potential adverse effects of the total inhibition of a specific miRNA remains to be carefully explored [76, 77].

G. Treatments directed at targeting metabolic hormone pathways Fibroblastic growth factor 21 (FGF21), secreted by the liver [78], is a metabolic regulator of glucose and lipid metabolism as well as energy homeostasis through signaling in the liver, the adipose tissue, and the hypothalamus via βKlotho and fibroblastic growth factor receptor [79]. Mechanisms underlying the pleiotropic effects of FGF21 include enhanced insulin sensitivity, reduced hepatic lipogenesis, induction of energy expenditure, and increased adiponectin secretion [79].

Liver (J Bajaj, Section Editor) Recently, elevated plasma levels of FGF21 were reported in patients with obesity and diabetes mellitus as well as in NAFLD [80, 81]. These observations together with the notion that potential differences between physiological FGF21 elevations and pharmacological effects of FGF21 likely exist [82] prompted a phase 1b proof-of-concept, randomized, placebo-controlled, and double-blind trial in obese patients with diabetes mellitus [83]. Patients received a subcutaneous injection of LY2405319, a FGF21 variant, and after 28 days of therapy, improvement of dyslipidemia, body weight, insulin, and adiponectin levels as well as fatty acid oxidation were found. Although some of the effects were only modest, further investigations need to explore the application of higher FGF21 doses for a longer period of time. As plasma FGF21 levels may substantially vary in humans [82], particularly in a heterogenous group such as metabolic disorders, certain baseline endogenous FGF21 levels can be identified that predict a more favorable response to FGF21 administration. Alternatively, FGF21 mimetics or bi-specific activators of the FGF21 pathway merit further exploration as well [84, 85]. Future studies also need to explore potential safety concerns such as bone mineral density loss described in rodents. Incretins such as glucagon-like peptide 1 are hormones that are released post-prandial by the gastrointestinal tract in response to bile acid activation of the G protein-coupled receptor TGR5 [86]. In addition to improving insulin sensitivity, glucagon-like peptide 1 appears to facilitate weight reduction by increasing satiety and slowing down gastrointestinal motility [87]. Additional effects of incretins include promoting fatty acid oxidation in the liver and inhibiting the release of fibroblast growth factor 21 [88]. Direct-acting glucagon-like peptide 1 analogue liraglutide (Victoza) and the synthetic receptor agonist exenatide (Byetta) have now been introduced in the treatment of diabetes mellitus and demonstrated a good safety profile [89, 90] supporting further exploration in patients with biopsy-proven NASH. A small study performed in Japan showed histological improvement of NASH after 24 weeks of treatment with liraglutide [91]. Results of a prospective, randomized, and placebo-controlled study enrolling 52 patients with biopsy-proven NASH, with and without diabetes mellitus, and treated with liraglutide for 48 weeks are eagerly awaited in early 2015.

H. Treatment targeting the intestinal microbiota It has become increasingly recognized that the gut-liver axis plays a key role in the development of metabolic disorders such as obesity and NAFLD, the intestinal microbiota being a key player in affecting metabolism of the host [92]. A key concept is that intestinal dysbiosis [93] promotes increased intestinal permeability thereby facilitating bacterial translocation with systemic upregulation of TNF-α [94, 95]. In addition, highly conserved motifs in pathogens termed pathogen-associated molecular patterns such as lipopolysaccharides from gut microbiota and saturated free fatty acids or damage-associated molecular patterns such as apoptotic DNA from damaged cells may reach the liver to activate pattern recognition receptors including toll-like receptors [96]. Within the liver, this results in an inflammatory response including production of proinflammatory cytokines and recruitment of immune cells. A small prospective proof-of-concept study with administration of Bifidobacterium longum with fructo-oligosaccharides demonstrated histological

Novel Therapies for NASH improvement of steatohepatitis without worsening of liver fibrosis [97]. Therefore, targeting the intestinal microbiota with pre- or probiotics or even fecal transplantation appears to be an interesting new avenue that needs further exploration. Clearly, more detailed knowledge about the microbiome in patients with NASH will greatly facilitate progress in this area of targeted therapy for NASH.

Conclusion Our understanding of how hepatic steatosis progresses to NASH has much improved over the last years and resulted in the identification of numerous potential targets for drug therapy. This resulted in an increased attention of biopharmaceutical companies to NASH, the most common metabolic liver disease with an enormous potential patient population. While the holy grail of NASH treatment is the regression of advanced fibrosis/cirrhosis, simply halting the progression would be clinically relevant. Numerous drug development programs for NASH are ongoing, most of them still in early phases. Nevertheless, the outlook is promising, and it is anticipated that the approval of new treatment options in the next years will drive the number of confirmed patients. Considering the pricing of oral drugs for diabetes therapy as comparable, the blockbuster potential is obvious. However, NASH will not be a winner-take-all market. The heterogeneity of the disease with different clinical phenotypes likely will be reflected by therapies that will combine two or more approved drugs.

Compliance with Ethics Guidelines Conflict of Interest Michael Fuchs has received honoraria from Intercept Pharmaceuticals.

Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by the author.

References and Recommended Reading Papers of particular interest, published recently, have been highlighted: • Of importance 1.

2. 3.

Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo clinic experiences with a hitherto unnamed disease. Mayo Clin Proc. 1980;55(7):434–8. Ong JP, Younossi ZM. Epidemiology and natural history of NAFLD and NASH. Clin Liver Dis. 2007;11(1):1–16. Lazo M, Clark JM. The epidemiology of nonalcoholic fatty liver disease: a global perspective. Semin Liver Dis. 2008;28(4):339–50.

4. 5.

6.

Sanyal AJ. NASH: a global health problem. Hepatol Res. 2011;41(7):670–4. Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013;58(3):593–608. Musso G, Gambino R, Cassader M, Pagano G. Metaanalysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-

Liver (J Bajaj, Section Editor) invasive tests for liver disease severity. Ann Med. 2011;43(8):617–49. 7. Söderberg C, Stal P, Askling J, Glaumann H, Lindberg G, Marmur J, et al. Decreased survival of subjects with elevated liver function tests during a 28-year-follow-up. Hepatology. 2010;51(2):595–602. 8. Wong JW, Cheung R, Ahmed A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology. 2014;59(6):2188– 95. 9. Perazzo H, Poynard T, Dufour JF. The interactions of nonalcoholic fatty liver disease and cardiovascular diseases. Clin Liver Dis. 2014;18(1):233–48. 10.• Sanyal AJ, Friedmann SL, McCullough AJ, Dimick L. Challenges and opportunities in drug and biomarker development for nonalcoholic steatohepatitis: findings and recommendations from an American Association for the Study of Liver Diseases (AASLD) – Food and Drug Administration (FDA) joint workshop. Hepatology 2014 [Epub ahead of print]. This work summarizes important considerations for clinical trial design in patients with NASH. 11. Perry RJ, Samuel VT, Petersen KF, Shulman GI. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature. 2014;510(7503):84–91. 12. Gruben N, Shiri-Sverdlov R, Koonen DP, Hofker MH. Nonalcoholic fatty liver disease: a main driver of insulin resistance or a dangerous liaison? Biochim Biophys Acta. 2014;1842(11):2329–43. 13. Choi SS, Diehl AM. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Curr Opin Lipidol. 2008;19(3):295–300. 14. Glass CK, Olefsky JM. Inflammation and lipid signaling in the etiology of insulin resistance. Cell Metab. 2012;15(5):635–45. 15. Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, et al. Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis. Hepatology. 2007;45(6):1366–74. 16. Leikin-Frenkel A, Goldiner I, Leikin-Gobbi D, Rosenberg R, Bonen H, Litvak A, et al. Treatment of preestablished diet-induced fatty liver by oral fatty acid-bile acid conjugates in rodents. Eur J Gastroenterol Hepatol. 2008;20(12):1205–13. 17. Safadi R, Konikoff FM, Mahamid M, Zelber-Sagi S, Halpern M, Gilat T, et al. The fatty acid-bile acid conjugate Aramchol reduces liver fat content in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2014;12912:2085–91. 18.• Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362D18]:1675–85. This study provides evidence for the use of vitamin E in select non-diabetic patients with NASH. 19. Klein EA, Thompson Jr IM, Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, et al. Vitamin E and the risk of

20. 21.

22.

23.

24.

25.

26.

27. 28.

29.

30.

31.

32. 33.

prostate cancer: the selenium and vitamin E cancer prevention trials (SELECT). JAMA. 2011;306(14):1549–56. Schurks M, Glynn RJ, Rist PM, Tzourio C, Kurth T. Effects of vitamin E on stroke subtypes: meta-analysis of randomized controlled trials. BMJ. 2010;341:c5702. Miller ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med. 2005;142(1):37–46. Scheen AJ. Pharmacodynamics, efficacy and safety of sodium-glucose cotransporter type 2 (SGLT2) inhibitors for the treatment of type 2 diabetes mellitus. Drugs. 2015;75(1):33–59. Sykes AP, Kemp GL, Dobbins R, O’Connor-Semmes R, Almond SR, Wilkison WO, et al. Randomized efficacy and safety trial of once-daily remogliflozin etabonate for the treatment of type 2 diabetes. Diabetes Obes Metab. 2015;17(1):98–101. Baeck C, Wehr A, Karlmark KR, Heymann F, Vucur M, Gassler N, et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. Gut. 2012;6193:416–26. Haukeland JW, Damas JK, Konopski Z, Loberg EM, Haaland T, Goverud I, et al. Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2. J Hepatol. 2006;44(6):1167–74. Kirovski G, Gaebele E, Dorn C, Moleda L, Niessen C, Weiss TS, et al. Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation. Int J Clin Exp Pathol. 2010;3(7):675–80. AASLD. Friedman. 2014. Wehr A, Baeck C, Ulmer F, Gassler N, Hittatiya K, Luedde T, et al. Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. PLoS One. 2014;9(11):e112327. Wehr A, Baeck C, Heymann F, Niemitz PM, Hammerich L, Martin C, et al. Chemokine receptor CXCR6-dependent hepatic NK T cell accumulation promotes inflammation and liver fibrosis. J Immunol. 2013;190(10):5226–36. Nieto N, Friedman SL, Cederbaum AI. Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells. J Biol Chem. 2002;277(12):9853– 64. Videla LA, Rodrigo R, Orellana M, Fernandez V, Tapia G, Quinones L, et al. Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients. Clin Sci. 2004;106(6):261–8. Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis. J Hepatol. 2001;35(2):297–306. Gambino R, Musso G, Cassader M. Redox balance in the pathogenesis of nonalcoholic fatty liver disease: mechanism and therapeutic opportunities. Antioxid Redox Signal. 2011;15(5):1325–65.

Novel Therapies for NASH 34.

Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology. 2001;120(5):1183–92. 35. Aoyama T, Paik YH, Watanabe S, Laleu B, Gaggini F, Fioraso-Cartier L, et al. Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent. Hepatology. 2012;56(6):2316–27. 36. Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schroeder K, et al. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/ NOX1 inhibitor in vivo. Free Radic Biol Med. 2012;53(2):289–96. 37. Luedde T, Kaplowitz N, Schwabe RF. Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterology. 2014;147(4):765–83. 38. Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, et al. Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology. 2003;125(2):437– 43. 39. Joka D, Wahl K, Moeller S, Schlue J, Vaske B, Bahr MJ, et al. Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis. Hepatology. 2012;55(2):455–64. 40. Witek RP, Stone WC, Karaca FG, Syn WK, Pereira TA, Agboola KM, et al. Pan-caspase inhibitor VX-166 reduces fibrosis in an animal model of nonalcoholic steatohepatitis. Hepatology. 2009;50(5):1421–30. 41.• Barreyro FJ, Holod S, Finocchietto PV, Camino AM, Aquino JB, Avagnina A et al. The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis n a murine model of non-alcoholic steatohepatitis. Liver Int. 2015;35(3):953-66. This study supports a role of pan-caspase inhibitors for NASH therapy. 42. Ratziu V, Sheikh MY, Sanyal AJ, Lim JK, Conjeevaram H, Chalasani N, et al. A phase 2, randomized, doubleblind, placebo-controlled study of GS-9450 in subjects with nonalcoholic steatohepatitis. Hepatology. 2012;55(2):419–28. 43. Shiffman ML, Pockros P, McHutchison JG, Schiff ER, Morris M, Burgess G. Clinical trial: the efficacy and safety of oral PF-03491390, a pancaspase inhibitor—a randomized placebo controlled study in patients with chronic hepatitis C. Aliment Pharmacol Ther. 2010;31(9):969–78. 44. Li L, Li J, Gao J. Functions of galectin-3 and its role in fibrotic disease. J Pharmacol Exp Ther. 2014;351(2):336–43. 45.• Traber GP, Zorner E. Therapy of experimental NASH and fibrosis with galectin inhibitors. PLoS One. 2013;8D12]:e83481. This study demonstrates galectin-3 inhibition as strategy for treatment of NASH.

46.

Traber GP, Chou H, Zomer E, Hong F, Klyosov A, Fiel MI, et al. Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease. PLoS One. 2013;8(10):e75361. 47. Harrison SA, Chalasani NP, Lawitz E, Marri S, Noureddin M, Sanyal AJ, et al. Early phase 1 clinical trial results of GR-MD-02, a galectin-3 inhibitor, in patients having non-alcoholic steatohepatitis (NASH) with advanced fibrosis. Hepatology. 2014;60:225A. 48. Carter EA, McCarron MJ, Alpert E, Isselbacher KJ. Lysyl oxidase and collagenase in experimental acute and chronic liver injury. Gastroenterology. 1982;82(3):526–34. 49. Moon HJ, Finney J, Ronnebaum T, Mure M. Human lysyl oxidase-like 2. Bioorg Chem. 2014;57:231–41. 50. Mallat A, Teixeira-Clerc F, Lotersztajn S. Cannabinoid signaling and liver therapeutics. J Hepatol. 2013;59(4):891–6. 51. Silvestri C, Di Marzo V. The endocannabinoid system in energy homeostasis and the etiopathology of metabolic disorders. Cell Metab. 2013;17(4):475–90. 52. Rossi F, Bellini G, Alisi A, Alterio A, Maione S, Perrone S, et al. Cannabinoid receptor type 2 functional variant influences liver damage in children with non-alcoholic fatty liver disease. PLoS One. 2012;7(8):e42259. 53. Chavez-Tapia NC, Tellez-Avila FI, Bedogni G, Croce LS, Masutti F, Tiribelli C. Systematic review and metaanalysis on the adverse events of rimonabant treatment: considerations for its potential use in hepatology. BMC Gastroenterol. 2009;9:75. 54. Fuchs M. Non-alcoholic fatty liver disease: the bile acid-activated farnesoid X receptor as an emerging treatment target. J Lipid. 2012;2012:934396. 55. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, Schoonjans K. Targeting bile-acid signaling for metabolic diseases. Nat Rev Drug Discov. 2008;7(8):678– 93. 56. Inagaki T, Moschetta A, Lee YK, Peng L, Zhao G, Downes M, et al. Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor. Proc Natl Acad Sci. 2006;103(10):3920–5. 57.• Neuschwandr-Tetri BA, Loomba R, Sanyal AJ, Lavine JE, van Natta ML, Abdelmalek MF et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicenter, randomized, placebo-controlled trial. The Lancet 2014 [Epub ahead of print]. This study confirms that targeting nuclear receptors is a viable option for treatment of NASH. 58.• Fang S, Suh JM, Reilly SM, Yu E, Osborn O, Lackey D et al. Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance. Nat Med. 2015;21(2):159-65. This work expands our knowledge of treatment options for metabolic disorders targeting organ-specific nuclear receptors. 59. Ryan KK, Tremaroli V, Clemmensen C, KovatchevaDatchary P, Myronovych A, Karns R, et al. FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature. 2014;509(7499):183–8.

Liver (J Bajaj, Section Editor) 60.

Fernandez-Miranda C, Perez-Carreras M, Colina F, Lopez-Alonso G, Vargas C, Solis-Herruzo JA. A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease. Dig Liver Dis. 2008;40(3):200–5. 61. Hoofnagle JH, Van Natta ML, Kleiner DE, et al. Vitamin E and changes in serum alanine aminotransferase levels in patients with non-alcoholic steatohepatitis. Aliment Pharmacol Ther. 2013;38:134–43. 62. Bojic LA, Huff MW. Peroxisome proliferator-activated receptor d: a multifaceted metabolic player. Curr Opin Lipidol. 2013;24(2):171–7. 63. Bays HE, Schwartz S, Littlejohn 3rd T, Kerzner B, Krauss RM, Karpf DB, et al. MBX-8025, a novel peroxisome proliferator receptor-delta agonist: lipid and other metabolic effects in dyslipidemic overweight patients with and without atorvastatin. J Clin Endocrinol Metab. 2011;96(9):2889–97. 64. Cariou B, Zair Y, Staels B, Bruckert E. Effects of the new dual PPARα/δ agonist GFT505 on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia or impaired glucose metabolism. Diabetes Care. 2011;34(9):2008–14. 65. Cariou B, Hanf R, Lambert-Porcheron S, Zair Y, Sauvinet V, Noel B, et al. Dual peroxisome proliferatoractivated receptor α/δ agonist GFT505 improves hepatic and peripheral insulin sensitivity in abdominally obese subjects. Diabetes Care. 2013;36(10):2923–30. 66.• Staels B, Rubenstrunk A, Noel B, Rigou G, Delataille P, Millatt LJ, et al. Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505 in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Hepatology. 2013;58D6]:1941–52. This study provides support for using GFT50 in patients with NASH. 67. Amir M, Czaja MJ. Autophagy in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol. 2011;5(2):159–66. 68. Kwanten WJ, Martinet W, Michielsen PP, Francque SM. Role of autophagy in the pathophysiology of nonalcoholic fatty liver disease: a controversial issue. World J Gastroenterol. 2014;20(23):7325–38. 69. Ding WX. Induction of autophagy, a promising approach for treating liver injury. Hepatology. 2014;59(1):1235–38. 70. Gonzalez-Rodriguez A, Mayoral R, Agra N, Valdecantos MP, Pardo V, Miquilena-Colina ME, et al. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFL D. Cell Death Dis. 2014;5:e1179. 71. Hsu CC, Schwabe RF. Autophagy and hepatic stellate cell activation—partners in crime? J Hepatol. 2011;55(6):1176–77. 72. Thoen LF, Guimaraes EL, Dolle L, Mannaerts I, Najimi M, Sokal E, et al. A role for autophagy during hepatic stellate cell activation. J Hepatol. 2011;55(6):1353–60. 73. Seok S, Fu T, Choi SE, Li Y, Zhu R, Kumar S, et al. Transcriptional regulation of autophagy by an FXRCREB axis. Nature. 2014;516(7529):108–11.

74. 75.

76. 77.

78.

79. 80.

81.

82. 83.

84.

85. 86.

87. 88.

89.

Lee JM, Wagner M, Xiao R, Kim KH, Feng D, Lazar MA, et al. Nutrient-sensing nuclear receptors coordinate autophagy. Nature. 2014;516(7529):112–5. Cheung O, Puri P, Eicken C, Contos MJ, Mirshahi F, Maher JW, et al. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression. Hepatology. 2008;48(6):1810–20. Rottiers V, Naar AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol. 2012;13(4):239–50. Hsu SH, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, ant-inflammatory, and antitumorigenic functions of miR-122 in live. J Clin Invest. 2012;122(8):2871–83. Markan KR, Naber MC, Ameka MK, Anderegg MD, Mangelsdorf DJ, Kliewer SA, et al. Circulating FGF21 is liver derived and enhances glucose uptake during refeeding and overfeeding. Diabetes. 2014;63(12):4057–63. Owen BM, Mangelsdorf DJ, Kliewer SA. Tissue-specific actions of the metabolic hormones FGF15/19 and FGF21. Trends Endocrinol Metab. 2015;26(1):22–9. Dushay J, Chui PC, Gopalakrishnan GS, Varela-Rey M, Crawley M, Fisher FM, et al. Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology. 2010;139(2):456–63. Dasarathy S, Yang Y, McCullough AJ, Marczewski S, Bennett C, Kalhan SC. Elevated hepatic fatty acid oxidation, high plasma fibroblast growth factor 21, and fasting bile acids in nonalcoholic steatohepatitis. Eur J Gastroenterol Hepatol. 2011;23(5):382–8. Angelin B, Larsson TE, Rudling M. Circulating fibroblast growth factor as metabolic regulators—a critical appraisal. Cell Metab. 2012;16(6):693–705. Gaich G, Chien JY, Fu H, Glass LC, Deeg MA, Holland WL, et al. The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Cell Metab. 2013;18(3):333–40. Smith R, Duguay A, Bakker A, Li P, Weiszmann J, Thomas MR, et al. FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/β-klotho bispecific protein. PLoS One. 2013;8(4):e61432. Smith R, Duguay A, Weiszmann J, Stanislaus S, Belouski E, Cai L, et al. A novel approach to improve the function of FGF21. BioDrugs. 2013;27(2):159–66. Thomas C, Gioiello A, Noriega L, Strehle A, Oury J, Rizzo G, et al. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab. 2009;10(3):167–77. Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology. 2007;132(6):2131–57. Olaywi M, Bhatia T, Anand S, Singhal S. Novel antidiabetic agents in non-alcoholic fatty liver disease: a mini-review. Hepatobiliary Pancreat Dis Int. 2013;12(6):584–8. Armstrong MJ, Houlihan DD, Rowe IA, Clausen WH, Elbrond B, Gough SC, et al. Safety and efficacy of liraglutide in patients with type 2 diabetes and elevated liver enzymes: individual patient data meta-analysis of

Novel Therapies for NASH

90.

91.

92. 93.

the LEAD program. Aliment Pharmacol Ther. 2013;37(2):234–42. Shao N, Kuang HY, Hao M, Gao XY, Lin WJ, Zou W. Benefits of exenatide on obesity and non-alcoholic fatty liver disease with elevated liver enzymes in patients with type 2 diabetes. Diabetes Metab Res Rev. 2014;30(6):521–9. Eguchi Y, Kitajima Y, Hyogo H, Takahashi H, Kojima M, Ono M et al. Pilot study of liraglutide effects in nonalcoholic steatohepatitis and non-alcoholic fatty liver disease with glucose intolerance in Japanese patients (LEAN-J). Heptology Res. 2015;45(3):269-78. Duseja A, Chawala YK. Obesity and NAFLD: the role of bacteria and microbiota. Clin Liver Dis. 2014;18(1):59–71. Mouzaki M, Comelli EM, Arendt BM, Bonengel J, Fung SK, Fisher SE, et al. Intestinal microbiota in patients with nonalcoholic fatty liver disease. Hepatology. 2013;58(1):120–7.

94.

95.

96. 97.

Wigg AJ, Roberts-Thompson IC, Dymock RB, McCarthy PJ, Grose RH, Cummins AG. The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumor necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis. Gut. 2001;48(2):206–11. Miele L, Valenza V, La Torre G, Montalto M, Cammarota G, Ricci R, et al. Increased permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology. 2009;49(6):1877–87. Bieghs V, Trautwein C. The innate immune response during liver inflammation and metabolic disease. Trends Immunol. 2013;34(9):446–52. Malaguarnera M, Vacante M, Antic T, Giordano M, Chisari G, Acquaviva R, et al. Bifidobacterium longum with fructo-oligosaccharides in patients with nonalcoholic steatohepatitis. Dig Dis Sci. 2012;57(2):545–53.