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Novel therapeutic targets for steatohepatitis夽 Arun J. Sanyal Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, MCV Box 980341, Richmond, VA 23298-0341, United States
Summary Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western hemisphere and is growing as an indication for liver transplantation. There are currently no approved therapies for NAFLD, especially its aggressive phenotype nonalcoholic steatohepatitis (NASH). However, there has been an explosion of information related to NASH that provides detailed data on the molecular pathogenesis of NASH and its progression to cirrhosis. The current review summarizes the pathophysiological rationale for the selection of specific targets for the treatment of NASH and provides an overview of the current strategies being used for the treatment of NASH and the pathophysiological rationale for the use of these strategies. Specifically, those targets that are being studied in both alcoholic and non-alcoholic steatohepatitis are also mentioned. © 2015 Published by Elsevier Masson SAS.
Introduction Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the most common causes of chronic liver disease worldwide. It has two principal phenotypes: non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH). NASH can progress to cirrhosis in 15—20% of subjects and is also a risk factor for hepatocellular cancer (HCC). The increasing
夽
This article is part of the special issue ‘‘Alcohol, Virus and Steatosis evolving to cancer’’ featuring the conference papers of the 10th International Symposium organized by the Brazilian Society of Hepatology in São Paulo, Brazil, September 30th—October 1st, 2015. E-mail address: [email protected]
incidence of HCC has been linked to the growing epidemic of obesity and NASH. By definition, NAFLD occurs in individuals who do not consume harmful amounts alcohol. However, a virtually identical histological phenotype can also develop in those with risk factors for NAFLD, e.g. obesity and the metabolic syndrome and who also consume more alcohol than allowed for the very strictly defined criteria for NAFLD. Recent studies have identified that, at a cellular level, similar pathophysiological pathways are frequently turned on in alcoholic and non-alcoholic steatohepatitis. This has led to growing interest in leveraging the substantial amount of literature developed for NASH to also treating alcoholic hepatitis. In this review, we will discuss the pathophysiological pathways being targeted for the treatment of steatohepatitis. The areas where it is relevant for alcoholic hepatitis, a specific note will be made.
http://dx.doi.org/10.1016/j.clinre.2015.05.012 2210-7401/© 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Sanyal AJ. Novel therapeutic targets for steatohepatitis. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.05.012
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Pathophysiological rationale for selection of therapeutic targets for steatohepatitis A core feature of steatohepatitis (SH) is predominantly macrovesicular steatosis. In addition, there is inflammation, hepatocyte ballooning and pericellular fibrosis. There is currently controversy whether a sequential progression from steatosis to steatohepatitis to cirrhosis occurs. While traditionally, steatosis was considered a non-progressive condition, recent data indicate that hepatic steatosis can progress to steatohepatitis and even advanced hepatic fibrosis [1,2]. There is therefore an emerging concept where the disease can be modeled from steatosis to steatohepatitis, which leads to progressive tissue remodeling resulting in cirrhosis. Genetics and how they may inform future therapy The PNPLA3 gene mutation was the first bonafide NASH related gene [3]. The presence of this gene mutation has been linked to increased steatosis, presence of steatohepatitis and more advanced disease. It has also been linked to alcoholic steatohepatitis [4]. It is over-represented in those of American Hispanic origin and under-represented in those of African American origin. It promotes but is not required for the development of steatohepatitis. Similarly, deep sequencing studies have identified the TM6SF2 as another NASH gene. Several other single nucleotide polymorphisms (SNPS) are also overexpressed in NASH. It is also important to remember that there are millions of SNPs in each individual and that SNPs do not always effect changes in physiology in isolation. We have recently shown that patterns of SNPs can cooperatively impact NASH phenotype and demonstrate a major role for cholesterol, bile acid metabolism related SNP pathways and multiple fibrosis related SNPs [5]. It is anticipated that in the future these will be related to the response to specific therapies and used to guide the choice of specific therapies. Systemic metabolic disturbances that contribute to steatohepatitis It has been known for at least two decades that NAFLD is associated with obesity, hypertension, type 2 diabetes mellitus, and dyslipidemia, the clinical hallmarks of the metabolic syndrome, which is characterized by insulin resistance (Table 1). Insulin resistance develops from macrophage infiltration into mainly visceral adipose tissue where it incites an inflammatory response and secretion of adipokines with a predominantly pro-inflammatory, profibrotic profile. These are further augmented by the acute phase reaction of the liver. Thus, both systemically and in the hepatic milieu, there is an excess of pro-inflammatory cytokines, such as TNF-␣ and IL-6. The metabolic consequence of this state is recognized as insulin resistance and is operationally defined by the ability to clear glucose from circulation at a given level of insulin. Insulin resistance is thus not a categorical state but rather a continuous variable. Several factors have been implicated in the initial genesis of adipose tissue inflammation, including relative ischemia and production of the hypoxia inducible factor-1, specific gut microflora and microflora-dependent inflammatory responses and hormones such as leptin [6]. These
Table 1 Systemic targets related to insulin resistance, inflammation and lipogensis. Intestinal microbiome- probiotics Microbiome derived endotoxin — Bovine colostrum with anti-endotoxin antibodies Hormones Adiponectin IGF Incretins Cytokines TNF-␣ IL-6 IL-8 Nuclear receptors PPAR-
demonstrate a key role for activation of the innate immune system in the development of insulin resistance, which is frequently associated with the presence of NASH. Several insulin sensitizers, such as GLP-1 agonists are currently study for the treatment of SH. Studies with metformin have been however disappointing for NASH [7]. TNF-␣, IL-6 and related gp130 related signaling, TLRs are all under investigation for the treatment of NASH. Another innovative approach is the use of bovine colostrum enriched with anti-endotoxin antibodies that are delivered orally to reduce the metabolic endotoxemia load, which drives activation of the innate immune system in the metabolic syndrome and NASH. This is currently under study both for alcoholic hepatitis and for non-alcoholic steatohepatitis. A novel metabolic pathway that could be potentially relevant for SH is the sodium glucose transporter (SGLT). Inhibition of renal Na glucose reabsorption by SGLT2 transporter has been shown to improve glycemic control and cause weight loss in diabetic subjects. Their potential role in the SH by mobilization of intrahepatic lipids to glucose that is then excreted by the kidneys is now undergoing evaluation. Development of hepatic steatosis and targets related mainly to steatosis. Hepatic steatosis develops when there is greater influx and production of lipids compared to their turnover and export (Table 2). Triglycerides (TG) are the principal form of lipids that accumulate in NASH. They are formed from influx of free fatty acids derived from increased peripheral lipolysis related to peripheral insulin resistance or from diet. They are also formed from de novo lipogenesis, which is increased in steatohepatitis. In alcoholic liver disease, there is a decrease in mitochondrial fatty acid oxidation. There is no convincing evidence that there is wide spread defect in triglyceride export in steatohepatitis and this is probably relevant in those with genetic disorders of triglyceride export e.g. abetalipoproteinemia. There is a multitude of signaling pathways that can contribute to increased lipid accumulation in the liver. SREBP-1c is a key transcriptional factor that promotes lipogenesis. There is also an increase in acyl coA carboxylase (ACC) and steroyl coA desaturase activity. These are currently being targeted as treatment for SH. Diacyl glycerol acyl transferase (DGAT) is a key enzyme required for the final step of TG synthesis and is also being targeted as a
Please cite this article in press as: Sanyal AJ. Novel therapeutic targets for steatohepatitis. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.05.012
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Novel therapeutic targets for steatohepatitis Table 2
Targets related to steatosis.
Cell surface receptors CB1 antagonists Gp130 Nuclear receptors LXR FXR PPAR-␣/␦ Transcriptional factors SREBP-1c HNF4 Lipogenic enzymes Acyl coA carboxylase Diacyl glycerol acyl transferase HMG CoA reductase Steroyl CoA desaturase Other lipids in liver Sphingosine-1-phosphate Ceramide Inflamamtory eicosanoids Phosphatidylcholine deficiency
therapeutic agent. Another important target to reduce steatosis is AMP kinase. This key enzyme regulates the phosphorylation of several key enzymes in the lipogenic pathway and also promotes mitochondrial uptake of fatty acids for oxidation [8]. Further upstream of the lipogenic pathways are other transcriptional networks, such as the mammalian target of rapamycin (mTOR) and a highly integrated set of orphan nuclear receptors, such as the Farnesoid X receptor and liver-X-receptor (LXR). Even further upstream of this are endocrine and paracrine mechanisms that can promote lipogenesis. Adiponectin is a key insulin sensitizer, which is often decreased in insulin resistant states [9]. Adiponectin is an adipokine and a PPAR- target. PPAR- has been used to treat SH but is limited by weight gain, fluid retention with congestive heart failure, increased osteopenia and risk of bladder cancer. Recently, a combination of PPAR-˛/ı agonism has been targeted for the treatment of SH [10]. PPAR-␣ agonists alone promote lipid oxidation and reduce steatosis in animal models [11]. The endocannabinoid system also promotes lipogenesis. Anandamide, the endogenous ligand for the cannabinoid receptor CB1 increased hepatic steatosis and lipogenic activity [12]. Initial attempts to block CB1 signaling to treat NASH failed due to off-target CNS effects including depression and increased suicide. Peripheral CB1 antagonists have now been developed and are actively being studied for NASH. The activity and amount of specific enzymes involved in lipogenesis are further modulated by several post-transcriptional mechanisms, including several miRNAs [13]. Currently mir122 and mir34a are being actively investigated as targets to reduced hepatic steatosis. In addition to TG, several other lipids accumulate in the liver in subjects with SH. These include free cholesterol, several sphingolipids, such as ceramide and sphingosine-1phosphate and various pro-inflammatory eicosanoids. There is an accompanying decrease in phospholipids, such as phospatidylcholine [14]. These lipids are highly active biologically and have myriad effects in promoting cell stress,
3 Table 3
Targets related to steatohepatitis.
Targets related to steatohepatitis Oxidative stress Glutathione SAMe JNK
apoptosis and inflammatory signaling. Unfortunately, to date, there is only some limited data with statins for decrease of cholesterol synthesis in NASH. The other lipid changes have not been sufficiently targeted for therapy but represent very attractive targets for therapy. Eicosanoids can activate NADPH oxidase and further modulate the hepatic metabolic response and inflammation; specific NADPH oxidase inhibitors are currently under development for the treatment of the metabolic syndrome and potentially for NASH. Mechanisms of steatohepatitis and related targets for therapy Several mechanisms have been implicated in the pathogenesis of cell injury in NASH (Table 3). These include free fatty acid induced cell toxicity (lipotoxicity), oxidative stress, endoplasmic reticulum stress (ER stress) and activation of the innate immune system, and cytokine-mediated cellular changes. Free fatty acids can cause cellular injury in several ways, including direct activation of inflammatory pathways, ER stress and activation of the innate immune system via Toll-like receptors. There are several potential sources of oxidative stress in NASH. NASH has been shown to be associated with mitochondrial injury and impairment of the electron transport system [15]. There is also activation of the cytochrome P450 2E1 with futile cycling, which leads to production of reactive oxygen species. There is also evidence for peroxisomal dysfunction in NASH [16]. A consequence of oxidative stress is activation of transcriptional factors, such as Nrf1, which turn on genetic pathways to limit injury due to oxidative stress. Oxidative stress leads to activation of inflammatory pathways, such as the JNK and NFB pathways. Glutathione is a major hepatic anti-oxidant and its turnover is increased under conditions of oxidative stress. Glutathione store repletion requires extracellular release of glutamyl-cysteine, which is taken up and converted to glutathione. Cystathionine serves as another source of cysteine and is linked to S-adenosyl homocysteine and S-adenosyl methionine (sAME). sAME and betaine are being used for the treatment of SH. Oxidative stress can deplete sAME and also affect gene expression via epigenetic modulation of the methylation status of DNA. Vitamin E is another agent that has been used for the treatment of SH and is an anti-oxidant [17]. Recently, a natural food supplement, bergamot, with anti-oxidant properties have been shown to improve liver enzymes in individuals at high risk for NAFLD [18]. Targets related to the inflammatory response in SH The hepatic inflammatory response to hepatocyte injury is believed to play an important role in driving the fibrogenic response and tissue remodeling that leads to cirrhosis and
Please cite this article in press as: Sanyal AJ. Novel therapeutic targets for steatohepatitis. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.05.012
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A.J. Sanyal Table 4
Targets related to inflammation.
Targets related to inflammation Inflammasome Toll-like receptors Chemokines
Table 5
Targets related to fibrosis.
Targets related to fibrosis Lysyl oxidase Galectin FGF-21 Connective tissue growth factor Insulin like growth factor
also sets the stage for hepatocellular cancer (Table 4). Several pathways for activation of the innate immune system have been implicated for development and perpetuation of the inflammatory response. Activation of the inflammasome has been linked to progressive liver injury in SH [19]. The inflammasome is controlled by IL-1; there are ongoing studies of specific IL-1 antagonists in both alcoholic and non-alcoholic steatohepatitis. The role of chemokines in driving inflammation in a variety of disease conditions is well established. Recently dual CCR2 and CXCR5 antagonist has been introduced in to clinical trials for NASH. By reducing the inflammatory response that continues to perpetuate the metabolic disturbances that drive steatosis, cell injury and further activation of inflammatory responses, it is hoped that this approach will reduce steatosis, hepatocyte injury, inflammation and eventually fibrosis. Unfortunately, other anti-inflammatory compounds have not been extensively evaluation. The use of anti-TNF antibodies (remicaide) in alcoholic hepatitis was associated with severe immunosuppression and increased mortality. Pentoxifylline, which also modulates TNF activity, has been shown to improve NASH in small pilot studies [20]. While it was believed to be useful for alcoholic hepatitis, a very large clinical trial has recently shown that it is not superior to placebo (data presented at AASLD 2014 but not yet published). A deuterated form of pentoxyfylline has been shown to preserve renal function in subjects with diabetes recently (personal communication from Concert Pharmaceuticals). It has not yet been studied for SH. Fibrosis related targets Many patients with SH come to medical attention when their disease is already quite advanced (Table 5). There is therefore a strong clinical rationale to target fibrosis in such patients. Fibrosis is linked to increased risk of HCC and increased mortality. Lysyl oxidase is an important target for reversal of fibrosis. It is a key enzyme involved in collagen cross-linking which stabilizes the collagen and reduces the ability of normal mechanisms to mobilize this key matrix protein. The levels of lysyl oxidase increase with increasing fibrosis and
recently have been shown to decrease following reversal of cirrhosis in those with hepatitis B treated with antiviral therapy. It has also been linked to promoting migration of tumor progenitors and contributing the development of HCC [21]. There are currently two major trials using a monoclonal antibody directed against lysyl oxidase for the treatment of NASH with bridging fibrosis or cirrhosis. Galectin has been linked to myocardial and hepatic fibrosis. Galectin has been targeted for the treatment of NASH with advanced fibrosis as well. Preliminary studies have confirmed its safety and tolerability. Phase 2 studies to clarify its efficacy are now under way. FGF-21 and connective tissue growth factor are also both implicated in SH related fibrogenesis. Both targets are currently under investigation for SH in phase 2A/2B studies. Preclinical data support an anti-fibrogenic role for these compounds. The emerging role of bile acids for the treatment of SH The last decade has seen much progress in the understanding of bile acids as key integrators of nutrient availability and the complex metabolic-inflammatory response to nutrient status. Bile acids can act through the FXR or TGR5 receptor system. Recently they have also been shown to act via the Sphingosine-1-phosphate kinase 2 (S1P2) receptors. Bile acids have multiple functions in the intestine, liver, and adipose tissue. They reduce intestinal inflammation and activation of the innate immune system in the colon via FXR. They also sensitize cells to insulin via FXR. Furthermore they decrease LXR activation and thus lipogenesis. They also have been shown to have anti-fibrotic effects. FXR agonists (INT-747) have been shown to improve all of the histological features of NASH and also improved hepatic fibrosis [22]. They are currently moving in to phase 3 trials for NASH. There is also an ongoing study of INT-747 for alcoholic hepatitis. TGR5 and S1P2 receptors also represent additional attractive targets for the treatment of the metabolic aspects of SH. The S1P2 pathway may be particularly relevant to activation of oncogenic signaling in SH.
Disclosure of interest Dr. Sanyal has stock options in Genfit. He has served as a consultant to AbbVie, Astra Zeneca, Nitto Denko, Nimbus, Salix, Tobira, Takeda, Fibrogen, Immuron, Exhalenz and Genfit. He has been an unpaid consultant to Intercept and Echosens. His institution has received grant support from Gilead, Salix, Tobira and Novartis.
Acknowledgement This review was written entirely by the authors without any external assistance. It is partly supported by a grant from the NIH to Dr. Sanyal T32 DK 007150-35.
References [1] Pais R, Pascale A, Fedchuck L, Charlotte F, Poynard T, Ratziu V. Progression from isolated steatosis to steatohepatitis and fibrosis in nonalcoholic fatty liver disease. Clin Res Hepatol Gastroenterol 2011;35:23—8.
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Novel therapeutic targets for steatohepatitis [2] McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM. Evidence of NAFLD progression from steatosis to fibrosingsteatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol 2015;62(5):1148—55, http://dx.doi.org/10.1016/j.jhep.2014.11.034 [PubMed PMID: 25477264. Epub 2014/12/06]. [3] Anstee QM, Day CP. The genetics of NAFLD. Nat Rev Gastroenterol Hepatol 2013;10:645—55. [4] Tian C, Stokowski RP, Kershenobich D, Ballinger DG, Hinds DA. Variant in PNPLA3 is associated with alcoholic liver disease. Nat Genet 2010;42:21—3. [5] Chen QR, Braun R, Hu Y, et al. Multi-SNP analysis of GWAS data identifies pathways associated with nonalcoholic fatty liver disease. PloS One 2013;8:e65982. [6] Nguyen TA, Sanyal AJ. Pathophysiology guided treatment of nonalcoholic steatohepatitis. J Gastroenterol Hepatol 2012;27(Suppl. 2):58—64. [7] Lavine JE, Schwimmer JB, Van Natta ML, et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. JAMA 2011;305:1659—68. [8] Crabb DW. Alcohol deranges hepatic lipid metabolism via altered transcriptional regulation. Trans Am Clin Climatol Assoc 2004;115:273—87. [9] Argentou M, Tiniakos DG, Karanikolas M, et al. Adipokine serum levels are related to liver histology in severely obese patients undergoing bariatric surgery. Obes Surg 2009;19: 1313—23. [10] Cariou B, Staels B. GFT505 for the treatment of nonalcoholic steatohepatitis and type 2 diabetes. Exp Opin Invest Drugs 2014;23:1441—8. [11] Harano Y, Yasui K, Toyama T, et al. Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, reduces hepatic steatosis and lipid peroxidation in fatty liver Shionogi mice with hereditary fatty liver. Liver Int 2006;26:613—20.
5 [12] Jeong WI, Osei-Hyiaman D, Park O, et al. Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver. Cell Metab 2008;7:227—35. [13] Cheung O, Puri P, Eicken C, et al. Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression. Hepatology 2008;48:1810—20. [14] Cramp DG. Lipid abnormalities in alcoholism. Contemp Issues Clin Biochem 1984;1:149—60. [15] Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120:1183—92. [16] Puri P, Wiest MM, Cheung O, et al. The plasma lipidomic signature of nonalcoholic steatohepatitis. Hepatology 2009;50:1827—38. [17] Sanyal AJ, Chalasani N, Kowdley KV, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. New Engl J Med 2010;362:1675—85. [18] Mollace V, Sacco I, Janda E, et al. Hypolipemic and hypoglycaemic activity of bergamot polyphenols: from animal models to human studies. Fitoterapia 2011;82:309—16. [19] Henao-Mejia J, Elinav E, Jin C, et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 2012;482:179—85. [20] Adams LA, Zein CO, Angulo P, Lindor KD. A pilot trial of pentoxifylline in nonalcoholic steatohepatitis. Am J Gastroenterol 2004;99:2365—8. [21] Wong CC, Tse AP, Huang YP, et al. Lysyl oxidase-like 2 is critical to tumor microenvironment and metastatic niche formation in hepatocellular carcinoma. Hepatology 2014;60:1645—58. [22] Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2015;385(9972): 956—65, http://dx.doi.org/10.1016/S0140-6736(14)61933-4 [PubMed PMID: 25468160. Epub 2014/12/04].
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Clinics and Research in Hepatology and Gastroenterology (2015) xxx, xxx—xxx
Available online at
ScienceDirect www.sciencedirect.com
MINI REVIEW
Novel therapeutic targets for steatohepatitis夽 Arun J. Sanyal Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, MCV Box 980341, Richmond, VA 23298-0341, United States
Summary Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western hemisphere and is growing as an indication for liver transplantation. There are currently no approved therapies for NAFLD, especially its aggressive phenotype nonalcoholic steatohepatitis (NASH). However, there has been an explosion of information related to NASH that provides detailed data on the molecular pathogenesis of NASH and its progression to cirrhosis. The current review summarizes the pathophysiological rationale for the selection of specific targets for the treatment of NASH and provides an overview of the current strategies being used for the treatment of NASH and the pathophysiological rationale for the use of these strategies. Specifically, those targets that are being studied in both alcoholic and non-alcoholic steatohepatitis are also mentioned. © 2015 Published by Elsevier Masson SAS.
Introduction Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the most common causes of chronic liver disease worldwide. It has two principal phenotypes: non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH). NASH can progress to cirrhosis in 15—20% of subjects and is also a risk factor for hepatocellular cancer (HCC). The increasing
夽
This article is part of the special issue ‘‘Alcohol, Virus and Steatosis evolving to cancer’’ featuring the conference papers of the 10th International Symposium organized by the Brazilian Society of Hepatology in São Paulo, Brazil, September 30th—October 1st, 2015. E-mail address: [email protected]
incidence of HCC has been linked to the growing epidemic of obesity and NASH. By definition, NAFLD occurs in individuals who do not consume harmful amounts alcohol. However, a virtually identical histological phenotype can also develop in those with risk factors for NAFLD, e.g. obesity and the metabolic syndrome and who also consume more alcohol than allowed for the very strictly defined criteria for NAFLD. Recent studies have identified that, at a cellular level, similar pathophysiological pathways are frequently turned on in alcoholic and non-alcoholic steatohepatitis. This has led to growing interest in leveraging the substantial amount of literature developed for NASH to also treating alcoholic hepatitis. In this review, we will discuss the pathophysiological pathways being targeted for the treatment of steatohepatitis. The areas where it is relevant for alcoholic hepatitis, a specific note will be made.
http://dx.doi.org/10.1016/j.clinre.2015.05.012 2210-7401/© 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Sanyal AJ. Novel therapeutic targets for steatohepatitis. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.05.012
+Model CLINRE-757; No. of Pages 5
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2
A.J. Sanyal
Pathophysiological rationale for selection of therapeutic targets for steatohepatitis A core feature of steatohepatitis (SH) is predominantly macrovesicular steatosis. In addition, there is inflammation, hepatocyte ballooning and pericellular fibrosis. There is currently controversy whether a sequential progression from steatosis to steatohepatitis to cirrhosis occurs. While traditionally, steatosis was considered a non-progressive condition, recent data indicate that hepatic steatosis can progress to steatohepatitis and even advanced hepatic fibrosis [1,2]. There is therefore an emerging concept where the disease can be modeled from steatosis to steatohepatitis, which leads to progressive tissue remodeling resulting in cirrhosis. Genetics and how they may inform future therapy The PNPLA3 gene mutation was the first bonafide NASH related gene [3]. The presence of this gene mutation has been linked to increased steatosis, presence of steatohepatitis and more advanced disease. It has also been linked to alcoholic steatohepatitis [4]. It is over-represented in those of American Hispanic origin and under-represented in those of African American origin. It promotes but is not required for the development of steatohepatitis. Similarly, deep sequencing studies have identified the TM6SF2 as another NASH gene. Several other single nucleotide polymorphisms (SNPS) are also overexpressed in NASH. It is also important to remember that there are millions of SNPs in each individual and that SNPs do not always effect changes in physiology in isolation. We have recently shown that patterns of SNPs can cooperatively impact NASH phenotype and demonstrate a major role for cholesterol, bile acid metabolism related SNP pathways and multiple fibrosis related SNPs [5]. It is anticipated that in the future these will be related to the response to specific therapies and used to guide the choice of specific therapies. Systemic metabolic disturbances that contribute to steatohepatitis It has been known for at least two decades that NAFLD is associated with obesity, hypertension, type 2 diabetes mellitus, and dyslipidemia, the clinical hallmarks of the metabolic syndrome, which is characterized by insulin resistance (Table 1). Insulin resistance develops from macrophage infiltration into mainly visceral adipose tissue where it incites an inflammatory response and secretion of adipokines with a predominantly pro-inflammatory, profibrotic profile. These are further augmented by the acute phase reaction of the liver. Thus, both systemically and in the hepatic milieu, there is an excess of pro-inflammatory cytokines, such as TNF-␣ and IL-6. The metabolic consequence of this state is recognized as insulin resistance and is operationally defined by the ability to clear glucose from circulation at a given level of insulin. Insulin resistance is thus not a categorical state but rather a continuous variable. Several factors have been implicated in the initial genesis of adipose tissue inflammation, including relative ischemia and production of the hypoxia inducible factor-1, specific gut microflora and microflora-dependent inflammatory responses and hormones such as leptin [6]. These
Table 1 Systemic targets related to insulin resistance, inflammation and lipogensis. Intestinal microbiome- probiotics Microbiome derived endotoxin — Bovine colostrum with anti-endotoxin antibodies Hormones Adiponectin IGF Incretins Cytokines TNF-␣ IL-6 IL-8 Nuclear receptors PPAR-
demonstrate a key role for activation of the innate immune system in the development of insulin resistance, which is frequently associated with the presence of NASH. Several insulin sensitizers, such as GLP-1 agonists are currently study for the treatment of SH. Studies with metformin have been however disappointing for NASH [7]. TNF-␣, IL-6 and related gp130 related signaling, TLRs are all under investigation for the treatment of NASH. Another innovative approach is the use of bovine colostrum enriched with anti-endotoxin antibodies that are delivered orally to reduce the metabolic endotoxemia load, which drives activation of the innate immune system in the metabolic syndrome and NASH. This is currently under study both for alcoholic hepatitis and for non-alcoholic steatohepatitis. A novel metabolic pathway that could be potentially relevant for SH is the sodium glucose transporter (SGLT). Inhibition of renal Na glucose reabsorption by SGLT2 transporter has been shown to improve glycemic control and cause weight loss in diabetic subjects. Their potential role in the SH by mobilization of intrahepatic lipids to glucose that is then excreted by the kidneys is now undergoing evaluation. Development of hepatic steatosis and targets related mainly to steatosis. Hepatic steatosis develops when there is greater influx and production of lipids compared to their turnover and export (Table 2). Triglycerides (TG) are the principal form of lipids that accumulate in NASH. They are formed from influx of free fatty acids derived from increased peripheral lipolysis related to peripheral insulin resistance or from diet. They are also formed from de novo lipogenesis, which is increased in steatohepatitis. In alcoholic liver disease, there is a decrease in mitochondrial fatty acid oxidation. There is no convincing evidence that there is wide spread defect in triglyceride export in steatohepatitis and this is probably relevant in those with genetic disorders of triglyceride export e.g. abetalipoproteinemia. There is a multitude of signaling pathways that can contribute to increased lipid accumulation in the liver. SREBP-1c is a key transcriptional factor that promotes lipogenesis. There is also an increase in acyl coA carboxylase (ACC) and steroyl coA desaturase activity. These are currently being targeted as treatment for SH. Diacyl glycerol acyl transferase (DGAT) is a key enzyme required for the final step of TG synthesis and is also being targeted as a
Please cite this article in press as: Sanyal AJ. Novel therapeutic targets for steatohepatitis. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.05.012
+Model CLINRE-757; No. of Pages 5
ARTICLE IN PRESS
Novel therapeutic targets for steatohepatitis Table 2
Targets related to steatosis.
Cell surface receptors CB1 antagonists Gp130 Nuclear receptors LXR FXR PPAR-␣/␦ Transcriptional factors SREBP-1c HNF4 Lipogenic enzymes Acyl coA carboxylase Diacyl glycerol acyl transferase HMG CoA reductase Steroyl CoA desaturase Other lipids in liver Sphingosine-1-phosphate Ceramide Inflamamtory eicosanoids Phosphatidylcholine deficiency
therapeutic agent. Another important target to reduce steatosis is AMP kinase. This key enzyme regulates the phosphorylation of several key enzymes in the lipogenic pathway and also promotes mitochondrial uptake of fatty acids for oxidation [8]. Further upstream of the lipogenic pathways are other transcriptional networks, such as the mammalian target of rapamycin (mTOR) and a highly integrated set of orphan nuclear receptors, such as the Farnesoid X receptor and liver-X-receptor (LXR). Even further upstream of this are endocrine and paracrine mechanisms that can promote lipogenesis. Adiponectin is a key insulin sensitizer, which is often decreased in insulin resistant states [9]. Adiponectin is an adipokine and a PPAR- target. PPAR- has been used to treat SH but is limited by weight gain, fluid retention with congestive heart failure, increased osteopenia and risk of bladder cancer. Recently, a combination of PPAR-˛/ı agonism has been targeted for the treatment of SH [10]. PPAR-␣ agonists alone promote lipid oxidation and reduce steatosis in animal models [11]. The endocannabinoid system also promotes lipogenesis. Anandamide, the endogenous ligand for the cannabinoid receptor CB1 increased hepatic steatosis and lipogenic activity [12]. Initial attempts to block CB1 signaling to treat NASH failed due to off-target CNS effects including depression and increased suicide. Peripheral CB1 antagonists have now been developed and are actively being studied for NASH. The activity and amount of specific enzymes involved in lipogenesis are further modulated by several post-transcriptional mechanisms, including several miRNAs [13]. Currently mir122 and mir34a are being actively investigated as targets to reduced hepatic steatosis. In addition to TG, several other lipids accumulate in the liver in subjects with SH. These include free cholesterol, several sphingolipids, such as ceramide and sphingosine-1phosphate and various pro-inflammatory eicosanoids. There is an accompanying decrease in phospholipids, such as phospatidylcholine [14]. These lipids are highly active biologically and have myriad effects in promoting cell stress,
3 Table 3
Targets related to steatohepatitis.
Targets related to steatohepatitis Oxidative stress Glutathione SAMe JNK
apoptosis and inflammatory signaling. Unfortunately, to date, there is only some limited data with statins for decrease of cholesterol synthesis in NASH. The other lipid changes have not been sufficiently targeted for therapy but represent very attractive targets for therapy. Eicosanoids can activate NADPH oxidase and further modulate the hepatic metabolic response and inflammation; specific NADPH oxidase inhibitors are currently under development for the treatment of the metabolic syndrome and potentially for NASH. Mechanisms of steatohepatitis and related targets for therapy Several mechanisms have been implicated in the pathogenesis of cell injury in NASH (Table 3). These include free fatty acid induced cell toxicity (lipotoxicity), oxidative stress, endoplasmic reticulum stress (ER stress) and activation of the innate immune system, and cytokine-mediated cellular changes. Free fatty acids can cause cellular injury in several ways, including direct activation of inflammatory pathways, ER stress and activation of the innate immune system via Toll-like receptors. There are several potential sources of oxidative stress in NASH. NASH has been shown to be associated with mitochondrial injury and impairment of the electron transport system [15]. There is also activation of the cytochrome P450 2E1 with futile cycling, which leads to production of reactive oxygen species. There is also evidence for peroxisomal dysfunction in NASH [16]. A consequence of oxidative stress is activation of transcriptional factors, such as Nrf1, which turn on genetic pathways to limit injury due to oxidative stress. Oxidative stress leads to activation of inflammatory pathways, such as the JNK and NFB pathways. Glutathione is a major hepatic anti-oxidant and its turnover is increased under conditions of oxidative stress. Glutathione store repletion requires extracellular release of glutamyl-cysteine, which is taken up and converted to glutathione. Cystathionine serves as another source of cysteine and is linked to S-adenosyl homocysteine and S-adenosyl methionine (sAME). sAME and betaine are being used for the treatment of SH. Oxidative stress can deplete sAME and also affect gene expression via epigenetic modulation of the methylation status of DNA. Vitamin E is another agent that has been used for the treatment of SH and is an anti-oxidant [17]. Recently, a natural food supplement, bergamot, with anti-oxidant properties have been shown to improve liver enzymes in individuals at high risk for NAFLD [18]. Targets related to the inflammatory response in SH The hepatic inflammatory response to hepatocyte injury is believed to play an important role in driving the fibrogenic response and tissue remodeling that leads to cirrhosis and
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A.J. Sanyal Table 4
Targets related to inflammation.
Targets related to inflammation Inflammasome Toll-like receptors Chemokines
Table 5
Targets related to fibrosis.
Targets related to fibrosis Lysyl oxidase Galectin FGF-21 Connective tissue growth factor Insulin like growth factor
also sets the stage for hepatocellular cancer (Table 4). Several pathways for activation of the innate immune system have been implicated for development and perpetuation of the inflammatory response. Activation of the inflammasome has been linked to progressive liver injury in SH [19]. The inflammasome is controlled by IL-1; there are ongoing studies of specific IL-1 antagonists in both alcoholic and non-alcoholic steatohepatitis. The role of chemokines in driving inflammation in a variety of disease conditions is well established. Recently dual CCR2 and CXCR5 antagonist has been introduced in to clinical trials for NASH. By reducing the inflammatory response that continues to perpetuate the metabolic disturbances that drive steatosis, cell injury and further activation of inflammatory responses, it is hoped that this approach will reduce steatosis, hepatocyte injury, inflammation and eventually fibrosis. Unfortunately, other anti-inflammatory compounds have not been extensively evaluation. The use of anti-TNF antibodies (remicaide) in alcoholic hepatitis was associated with severe immunosuppression and increased mortality. Pentoxifylline, which also modulates TNF activity, has been shown to improve NASH in small pilot studies [20]. While it was believed to be useful for alcoholic hepatitis, a very large clinical trial has recently shown that it is not superior to placebo (data presented at AASLD 2014 but not yet published). A deuterated form of pentoxyfylline has been shown to preserve renal function in subjects with diabetes recently (personal communication from Concert Pharmaceuticals). It has not yet been studied for SH. Fibrosis related targets Many patients with SH come to medical attention when their disease is already quite advanced (Table 5). There is therefore a strong clinical rationale to target fibrosis in such patients. Fibrosis is linked to increased risk of HCC and increased mortality. Lysyl oxidase is an important target for reversal of fibrosis. It is a key enzyme involved in collagen cross-linking which stabilizes the collagen and reduces the ability of normal mechanisms to mobilize this key matrix protein. The levels of lysyl oxidase increase with increasing fibrosis and
recently have been shown to decrease following reversal of cirrhosis in those with hepatitis B treated with antiviral therapy. It has also been linked to promoting migration of tumor progenitors and contributing the development of HCC [21]. There are currently two major trials using a monoclonal antibody directed against lysyl oxidase for the treatment of NASH with bridging fibrosis or cirrhosis. Galectin has been linked to myocardial and hepatic fibrosis. Galectin has been targeted for the treatment of NASH with advanced fibrosis as well. Preliminary studies have confirmed its safety and tolerability. Phase 2 studies to clarify its efficacy are now under way. FGF-21 and connective tissue growth factor are also both implicated in SH related fibrogenesis. Both targets are currently under investigation for SH in phase 2A/2B studies. Preclinical data support an anti-fibrogenic role for these compounds. The emerging role of bile acids for the treatment of SH The last decade has seen much progress in the understanding of bile acids as key integrators of nutrient availability and the complex metabolic-inflammatory response to nutrient status. Bile acids can act through the FXR or TGR5 receptor system. Recently they have also been shown to act via the Sphingosine-1-phosphate kinase 2 (S1P2) receptors. Bile acids have multiple functions in the intestine, liver, and adipose tissue. They reduce intestinal inflammation and activation of the innate immune system in the colon via FXR. They also sensitize cells to insulin via FXR. Furthermore they decrease LXR activation and thus lipogenesis. They also have been shown to have anti-fibrotic effects. FXR agonists (INT-747) have been shown to improve all of the histological features of NASH and also improved hepatic fibrosis [22]. They are currently moving in to phase 3 trials for NASH. There is also an ongoing study of INT-747 for alcoholic hepatitis. TGR5 and S1P2 receptors also represent additional attractive targets for the treatment of the metabolic aspects of SH. The S1P2 pathway may be particularly relevant to activation of oncogenic signaling in SH.
Disclosure of interest Dr. Sanyal has stock options in Genfit. He has served as a consultant to AbbVie, Astra Zeneca, Nitto Denko, Nimbus, Salix, Tobira, Takeda, Fibrogen, Immuron, Exhalenz and Genfit. He has been an unpaid consultant to Intercept and Echosens. His institution has received grant support from Gilead, Salix, Tobira and Novartis.
Acknowledgement This review was written entirely by the authors without any external assistance. It is partly supported by a grant from the NIH to Dr. Sanyal T32 DK 007150-35.
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