Nonalcoholic Steatohepatitis (NASH)

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1 PSH Clinical Guidelines Statement 2017 Nonalcoholic Steatohepatitis (NASH) Dr. Adil Naseer Khan Associate Professor of Gastroenterology Ayub Medical College Abbottabad Non-alcoholic fatty liver disease (NAFLD) is defined, as excess accumulation of fat in the liver. 1 NAFLD has become the most common cause for chronic liver disease. NAFLD has two subtypes distinguished histopathologically with different prognosis: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Non-alcoholic steatohepatitis (NASH) is a subset of NAFLD, estimated to affect 2 5% of Americans, in which increased liver fat is accompanied by cellular injury, inflammatory infiltrate and, subsequently, liver fibrosis, which can progress to cirrhosis with its associated complications. 2 The diagnosis of NAFLD is made after excluding both secondary causes and of a daily alcohol consumption of <30 g for men and <20 g for women. Alcohol consumption above these limits indicates alcoholic liver disease.. The definitive diagnosis of NASH requires a liver biopsy. NAFLD has association with metabolic syndrome. All individuals with steatosis should be screened for features of Metabolic syndrome independent of liver enzymes.components of metabolic syndrome are: Waist circumference >94/>80 cm for men/women. Arterial pressure >130/85 mmhg or treated for hypertension. Fasting glucose >100 mg/dl (5.6 mmol/l) or treated for T2DM Serum triacylglycerols >150 mg/dl (>1.7 mmol/l). Low HDL cholesterol Not necessarily NAFLD may be associated with all the components of metabolic syndrome. NAFLD is also present in 7% of normalweight (lean) persons. NAFLD is associated with weight gain, western diet, high fructose intake, sugar sweetened beverages and saturated fats consumptions. Several genetic modifiers of NAFLD have been identified but a minority has been robustly validated. 4 Carrier of PNPLA 3 and TM6SF2 have increased risk of NASH. Two subtypes of NAFLD i.e. NAFL and NASH can only be distinguished histologically. NAFL encompasses: a) steatosis alone, b) steatosis with lobular or portal inflammation, without ballooning, or c) steatosis with ballooning but without inflammation. The diagnosis of NASH requires the joint presence of steatosis, ballooning and lobular inflammation. NAFLD is tightly associated with Insulin resistance not only in the liver, but also in muscle and adipose tissues, and also with the metabolic syndrome. Importantly, patients

2 with BMI <30 kg/m2 (or even <25 kg/m2) but with visceral fat accumulation or dysfunctional adipose tissue can exhibit NAFLD with/without abnormal liver enzyme. 5 NAFLD has association with coronary artery disease and its incidence is high as compared to matched controls. CVD is a more common cause of death than liver disease in NAFLD.It is recommended that all patients with NAFLD should be screened for underlying coronary artery disease and conversely patient with coronary artery disease should be screened for NAFLD. NAFLD is also associated with risk of HCC. The cumulative incidence of NAFLD-associated HCC >10-fold higher in T2DM and obesity Chronic kidney disease (CKD) can be found in 20-50% of NAFLD patients, particularly in biopsy-proven NASH. 6 NAFLD is also associated with colorectal cancer, metabolic bone disease (vitamin D deficiency, osteoporosis) Pathogenesis of nonalcoholic fatty liver disease Nonalcoholic fatty liver disease (NAFLD) is a clinico-histopathological entity with histological features that resemble alcoholinduced liver injury, but by definition, it occurs in patients with little or no history of alcohol consumption. It encompasses a histological spectrum that ranges from fat accumulation in hepatocytes without concomitant inflammation or fibrosis (simple hepatic steatosis) to hepatic steatosis with a necroinflammatory component (steatohepatitis) that may or may not have associated fibrosis. The latter condition, referred to as nonalcoholic steatohepatitis (NASH), may progress to cirrhosis in up to 20 percent of patients. 7 NASH is now recognized to be a leading cause of cryptogenic cirrhosis. 8 Still exact pathogenesis of NAFLD/NASH is not fully understood. Insulin resistance is considered most important component of its pathogenesis leading to hepatic steatosis and may be steatohepatitis. Some researchers are of the opinion that there is second hit or oxidative stress injury in patients manifesting progressive disease i.e. NASH CAUSES OF TRIGLYCERIDE ACCUMULATION Triglyceride accumulation in the liver can occur from 1) The excessive importation of free fatty acids (FFA) from adipose tissue, 2) Diminished hepatic export of FFA (secondary to reduced synthesis or secretion of very low-density lipoprotein [VLDL]) 3) Impaired beta-oxidation of FFA. In obesity and rapid weight loss increased delivery of triglycerides leads to excessive importation of FFA in liver similarly conversion of carbohydrates and protein in overfeeding and total parenteral nutrition. Abetalipoproteinemia, protein malnutrition, or choline deficiency are associated with impaired VLDL synthesis. Patients with nonalcoholic steatohepatitis (NASH) may have a defect in postprandial ApoB secretion, leading to triglyceride accumulation. Amiodarone and tetracycline by inhibition of microsomal triglyceride transfer protein (MTP) impairs the lipidation of APO B leading to triglyceride deposition in liver. Vitamin B5 (pantothenic acid) deficiency, excessive alcohol consumption, or coenzyme A deficiency (as can occur with valproic acid or chronic aspirin use) can lead to Impaired

3 beta-oxidation of FFA to adenosine triphosphate (ATP). Activation of peroxisome proliferator-activated receptor alpha appears to have a central role in stimulating betaoxidation and disposing hepatic fatty acids in NASH. Administration of adiponectin improved hepatomegaly, steatosis, and alanine aminotransferase levels in obese, leptin deficient mice. Also implicated in the steatosis pathway is the cannabinoid receptor type 1 (CB1). Administration of a CB1 receptor antagonist rapidly abolished hepatic steatosis, improved aminotransferase levels, reduced the levels of proinflammatory cytokines, and increased adiponectin levels in leptin-deficient mice. INSULIN RESISTANCE Insulin resistance has a major role in NAFLD/NASH pathogenesis. Insulin resistance has also been documented in lean patients with NASH. Not in all patients with NASH insulin resistance is established so NASH is a heterogeneous syndrome which may be having more than one causes. Insulin resistance results enhanced peripheral lipolysis, increased triglyceride synthesis and increased hepatic uptake of fatty acids. This results in shift from carbohydrate to FFA beta oxidation. IRON insulin resistance leads to increase hepatic iron levels and improved glycemic control improves serum ferritin and hepatic iron levels. LEPTIN Leptin is a peptide produced primarily in adipose tissue. Decreased or absent levels of leptin are associated with obesity. Leptin may have a role in inducing fibrosis in NASH. Leptin induces dephosphorylation of insulin-receptor substrate 1, rendering hepatocytes more insulin-resistant. Blood leptin levels correlate with the degree of fibrosis in patients with chronic hepatitis C. 9 ADIPONECTIN A hormone produced by adipose tissue. It has got desirable effects on lipid clearance from plasma and beta oxidation of fatty acids in muscles. In addition, it has got anti-inflammatory effects, suppressing TNFalpha production in the liver. Low adiponectin levels have been associated with NAFLD/fibrosis/metabolic syndrome. RESISTIN Resistin is an adipose-derived protein is implicated in the of insulin resistance. Overexpression of resistin in a mouse model led to glucose intolerance, hyperinsulinemia, and impaired suppression of free fatty acid levels INTESTINAL MICROBES small intestinal bacterial overgrowth was observed significantly more often in patients with nonalcoholic steatohepatitis (NASH) compared with controls. 10 Investigations Mostly hepatic steatosis is discovered incidentally while patient undergoing workup for some other reason because NAFLD/NASH does not have specific symptoms. The incidental discovery of steatosis should lead to comprehensive evaluation of family and personal history of NAFLD associated diseases and the exclusion of secondary causes of steatosis. Metabolic work-up has to include a careful assessment of all components of Metabolic syndrome. Similarly, the presence of obesity/ T2DM or the incidental finding of raised liver enzymes in patients with metabolic risk factors should prompt non-invasive screening to predict steatosis, NASH and fibrosis.

4 In persons with NAFLD, screening for diabetes is mandatory, by fasting or random blood glucose or HbA1c and if available by the standardized 75 g OGTT in high-risk groups. Surrogate markers of fibrosis (NFS, FIB-4 or FibroTest) should be calculated for every NAFLD patient, in order to rule out significant fibrosis. If significant fibrosis cannot be ruled out, patients should be referred to a Liver Clinic for transient elastography; if significant fibrosis is confirmed, the final diagnosis should be made by liver biopsy. In patient with NAFLD detailed history including personal or family history of DM, hypertension, CVD, alcohol intake with documentation of amount taken should be asked. Drugs causing hepatic steatosis should be asked followed by examination including weight and BMI. Following set of biochemical investigations should be carried out in all the patients with initial diagnosis of NAFLD Hepatitis B/Hepatitis C virus infection Liver enzymes (aspartate and alanine transaminases (γ-glutamyl-trans-peptidase)) Fasting blood glucose, HbA1c, OGTT, (fasting insulin [HOMA-IR]) Complete blood count Serum total and HDL-cholesterol, triacylglycerol, uric acid Ultrasonography (if suspected for raised liver enzymes) Extended 1. Ferritin and transferrin saturation 2. Tests for coeliac and thyroid diseases, polycystic ovary syndrome 3. Tests for rare liver diseases (Wilson, autoimmune disease, α1-antitrypsin deficiency) US has limited sensitivity and does not reliably detect steatosis when <20% 11 or in individuals with high body mass index (BMI) (>40 kg/m2). 12 Despite observer dependency, US robustly diagnoses moderate and severe steatosis and provides additional hepatobiliary information, hence it should be performed as a first-line diagnostic test Next step after initial evaluation would be whether patient is having simple hepatic steatosis/nafl or NASH. Gold standard is liver biopsy to establish the diagnosis of NASH. Because of its limitations of being invasive and not well coming by patients cannot be carried out in all the patients with initial diagnosis of NAFLD. Different noninvasive serological and radiological set of investigation have been developed to identify patient with progressive disease which requires specific intervention. These noninvasive markers have been validated in different studies. NASH has to be diagnosed by a liver biopsy showing steatosis, hepatocyte ballooning and lobular inflammation. Noninvasive assessment of hepatic fibrosis: Overview of serologic and radiographic tests STAGES OF FIBROSIS Noninvasive tests of hepatic fibrosis attempt to predict the stage of hepatic fibrosis that would be seen histologically. There are several histologic scoring systems for chronic liver disease. Many use five-point scales such as the METAVIR score. F0: No fibrosis F1: Portal fibrosis without septa F2: Few septa F3: Numerous septa without cirrhosis

5 F4: Cirrhosis Patients are typically considered to have significant fibrosis if their fibrosis score is F2. Noninvasive tests of hepatic fibrosis are primarily used to differentiate patients with significant fibrosis (F2 to F4) from those with minimal or no fibrosis (F0 to F1). Serologic panels of tests and radiologic tests are non invasive modalities for fibrosis. When available both should be used to augment the results. Serologic markers of hepatic fibrosis can broadly be categorized as indirect or direct: Direct markers of fibrosis reflect extracellular matrix turnover. Examples include procollagen types I and III, hyaluronic acid, and tissue inhibitor of metalloproteinase Indirect markers reflect alterations in hepatic function, but do not directly reflect extracellular matrix metabolism. Examples include the platelet count, coagulation studies, and liver aminotransferases. Direct serum Markers There are four commercial serum marker systems that have been extensively validated: 1. FibroTest/FibroSure (marketed in the United States by LabCorp), 2. Hepascore (Quest Diagnostics), 3. FibroSpect (Prometheus Corp), and the 4. European Liver Fibrosis Study Group panel. All the serum tests have limitations: They typically reflect the rate of matrix turnover, not deposition, and thus tend to be more elevated when there is high inflammatory activity. By contrast, extensive matrix deposition can go undetected if there is minimal inflammation. None of the markers are liver-specific, and concurrent sites of inflammation may contribute to serum levels. Serum levels are affected by clearance rates, which may be impaired either due to sinusoidal endothelial cell dysfunction or impaired biliary excretion. They are surrogates, not biomarkers. Indirect Markers Aspartate aminotransferase to platelet ratio (APRI) has also been studied extensively. The APRI has the advantage of being easily calculated using data available from routine laboratory tests AST to platelet ratio index The APRI is based on the AST level and platelet count and is easy to calculate]. The APRI is calculated using the AST elevation (which is the AST level divided by the upper limit of normal [ULN] for the lab) and the platelet count per mm3 divided by APRI = (AST elevation/platelet count) x 100 For predicting significant fibrosis (F2 to F4), an APRI cutoff of 0.7 had a sensitivity of 77 percent and a specificity of 72 percent. For predicting cirrhosis (F4), an APRI cutoff of 1.0 had a sensitivity of 76 percent and a specificity of 72 percent AST/ALT ratio The AST/ALT ratio is approximately 0.8 in normal subjects. Some studies have suggested that a ratio >1 suggests the presence of cirrhosis. However, study findings have been inconsistent, and the clinical utility of this ratio for diagnosis of cirrhosis remains uncertain FIB-4 index The FIB-4 index combines biochemical values (platelet count, ALT, and AST) and age. it performed better than other

6 serologic markers for predicting advanced fibrosis in patients with NAFLD. 13 NAFLD fibrosis score The NAFLD fibrosis score is another score used to assess the probability of fibrosis in patients with NAFLD. It takes into account the patient's age, body mass index (BMI), blood glucose levels, aminotransferase levels, platelet count, and albumin. In a validation study, a high NAFLD fibrosis score cutoff (>0.676) was associated with a positive predictive value for advanced fibrosis (F3 to F4) of 82 percent (sensitivity 43 percent, specificity 96 percent), and a low cutoff value (<-1.455) was associated with a negative predictive value of 88 percent (sensitivity 77 percent, specificity 71 percent). 14 IMAGING TESTS Radiologic methods for assessing hepatic fibrosis include ultrasound-based transient elastography, magnetic resonance elastography, acoustic radiation force impulse imaging, and cross-sectional imaging. Ultrasound-based transient elastography is the most widely used. IMAGING TESTS Radiologic methods for staging hepatic fibrosis are emerging as promising tools. The methods include ultrasound-based transient elastography, magnetic resonance elastography (MRE), acoustic radiation force impulse imaging (ARFI), and cross-sectional imaging Ultrasound-based elastography Ultrasound-based elastography rapidly and noninvasively measures mean hepatic tissue stiffness. Ultrasound-based elastography fulfills many features desirable of noninvasive assessment of hepatic fibrosis; it is quick, inexpensive, reproducible, painless, and examines a large mass of liver tissue, thereby reducing sampling error. Magnetic resonance elastography Elastography can also be performed with magnetic resonance imaging (MRI). The technique involves placing a probe against the patient's back. The probe emits low-frequency vibrations that pass through the liver and can be measured by the MRI spin echo sequence. MRE is available in the United States. A meta-analysis that included 12 studies of MRE found the following test characteristics: Detecting any fibrosis (F 1): Optimal cutoff 3.45 kpa, sensitivity 73 percent, specificity 79 percent Detecting significant fibrosis ( F2): Optimal cutoff 3.66 kpa, sensitivity 79 percent, specificity 81 percent Detecting advanced fibrosis ( F3): Optimal cutoff 4.11 kpa, sensitivity 85 percent, specificity 85 percent Detecting cirrhosis (F4): Optimal cutoff 4.71, sensitivity 91 percent, specificity 81 percent Acoustic radiation force impulse imaging ARFI imaging is another ultrasound-based approach for estimating liver stiffness. ARFI imaging uses short-duration, high-intensity acoustic pulses to produce mechanical excitation in tissue. This results in localized tissue displacement and shear wave propagation. The velocity of these waves correlates with the degree of fibrosis (the shear wave velocity increases as the amount of fibrosis increases), though optimal cutoff values vary among studies. Real-time shear wave elastography (SWE) SWE is a technique that is based on the same principles as ultrasound-based transient elastography. SWE estimates the speed of a shear wave through the liver to measure liver stiffness

7 Treatment of NAFLD Since last one-decade extensive research has been carried out to understand the pathophysiology of NAFLD. This led to different therapies in the treatment of NAFLD/NASH with variable results. They can be groped broadly into four classes, according to areas of their action at different level. (1) Medications that decreases hepatic fat accumulation and metabolic stress. (2) Medications ameliorating oxidative stress /inflammation /injury components of NASH. (3) Medications altering interaction between the gut and the liver (4) Medications targeting fibrosis In general, NAFLD is a slowly progressive disease, both in adults and in children, but fibrosis rapidly progresses in 20% of cases. 15 MEDICATIONS WITH A PRIMARY METABOLIC TARGET PPAR agonists Peroxisome proliferator-activator receptors (PPARs) are a family of nuclear receptors that bind fatty acids and their derivatives and regulate a wide range of metabolic processes. There are three PPARs α, β/δ and γ. PPARα has wide distribution in the liver, adipose tissue, heart, skeletal muscle and kidney. Fibrates a synthetic agonist of PPARα, have an established role in the treatment of hypertriglyceridemia, but have not shown a consistent beneficial effect for the treatment of NAFLD. 16 PPARδ, another member of the PPAR family expressed in liver, skeletal muscle and macrophages. Their activation has effects on insulin sensitivity, hepatic glucose production, fatty acid oxidation and macrophage and Kupffer cell activation. Due to safety concerns further studies on PPARδ agonist are halted but they significantly decreased hepatic fat contents in their pilot study. Elafibranor is a dual PPARα/δ agonist, aiming to combine the beneficial effects of activating the two receptors. A study is in progress in pts with NAS 4, to see the effects of 72 weeks of treatment with 120 mg/day on NASH resolution. Thiazolidinediones (TZDs), synthetic PPARγ agonists, are insulin sensitizers with proven efficacy for treatment of diabetes and have been shown in multiple trials to be effective for the treatment of NASH. In the PIVENS RDBPCT, the largest trial of a PPARγ agonist to date, pioglitazone 30 mg/day for 96 weeks showed histological improvement in 34% of subjects compared with 19% of controls. The glitazars dual PPARα/γ agonists, aiming to combine the beneficial effects of PPARα and PPARγ agonism. However, development of most compounds in this class has been halted due to safety concerns. Saroglitazar, is currently approved in India for the treatment of diabetic dyslipidemia A retrospective analysis of patients with NAFLD treated with saroglitazar for dyslipidemia demonstrated significant and marked decrease in ALT (from 64±6 to 28±3, p<0.01) after 24 weeks of treatment. 17 Targeting the FXR-bile acid axis The interaction of bile acids with the farnesoid X receptor (FXR), negatively regulates bile acid synthesis and decrease hepatic lipogenesis and steatosis, hepatic gluconeogenesis and improve peripheral insulin sensitivity. Obeticholic acid (6-ethylchenodeoxycholic acid (OCA)) is a synthetic lipophilic bile acid acting as FXR. In FLINT study obetocholic

8 acids showed significant improvement in histology, fibrosis score and liver enzymes. Inhibition of de novo lipogenesis Aramchol is a conjugate of cholic and arachidic acid that was shown to inhibit stearoyl CoA desaturase (SCD) and de novo lipogenesis in cell and animal models. Incretins and DPP-4 inhibitors Glucagon-like peptide 1 (GLP-1) is an incretin secreted by intestinal L-cells in response to meal ingestion and acts on the pancreas to improve glycemic control, insulin secretion, inhibiting α-cell glucagon secretion. Incretins also enhances insulin sensitivity and glycogen synthesis.in LEAN study in comparison to placebo liraglutide treatment has shown significant improvement in histology of biopsy proven NASH. Dipeptidyl peptidase 4 (DPP-4) increases the effect of incretin by inhibiting the enzyme responsible for their rapid degradation.in randomized controlled trials they have not shown any significant role in management of NASH. Lipid-lowering agents In small clinical trials statins have shown promising results in improving histology in NASH but whether these results can be replicated in large clinical trial is yet to be seen. Ezetimibe has sown no clinical and histological benefit in NASH. MEDICATIONS AFFECTING OXIDATIVE STRESS AND INFLAMMATION Antioxidants In the PIVENS phase III RDBPCT vitamin E at a dose of 800 IU/day for 96 weeks was superior to placebo, achieving histological response in 43% of subjects (compared with 19% of placebo, p=0.001) and resolution of NASH in 36%. Cysteamine is an aminothiol that can act as scavenger for reactive oxygen species and can replenish glutathione stores, and thus has potential benefit in NASH. Targeting apoptosis and TNFα pathway Emricasan is an oral irreversible pan-caspase inhibitor with high first-pass metabolism, making it a potentially attractive agent for the treatment of liver diseases associated with apoptosis ENCORE-NF, an ongoing phase IIb trial is evaluating role of emricasan in NASH. Pentoxifylline because of its anti TNF and antioxidative effect may have potential role in treatment of NASH and is under evaluation in different trials Immune modulators Two IκB kinases (IKKs) IKKε and TBK1 in animals with diet induced obesity they are upregulated in adipose tissues and deletion has protective effects on insulin resistance, obesity and steatosis Amlexanox, a medication used to treat asthma and aphthous ulcers, was found in a screening assay to be an inhibitor of both IKKε and TBK1. A phase II RDBPCT is currently assessing the effects of 12 weeks of amlexanox in patients with diabetes, obesity and fatty liver on hepatic fat content. Cenicriviroc is an oral antagonist of CCR2/ CCR5, the chemokine receptors for MCP1 and RANTES respectively which are inflammatory chemokines and have role in migration of inflammatory cells in to the liver. CENTAUR, an ongoing phase IIb trial, is assessing the histological effects of up to 2 years of cenicriviroc or placebo on patients with NASH and fibrosis (but not cirrhosis).

9 MEDICATIONS WITH A PRIMARY GUT TARGET Antiobesity medications Lifestyle modification and weight loss is the back bone of therapeutic intervention in treatment of NASH. Orlistat does not have any independent effect in management of NASH but indirectly can help by weight reduction Targeting gut microbiome The effects of altering gut microbiota on liver have been studied. Insulin sensitivity was shown to improved by transplanting FMT from lean subjects to person with metabolic syndrome. In an ongoing open-label pilot study, investigators are performing FMT from lean donors to patients with biopsy-proven noncirrhotic NASH to assess the effects on liver fat content and markers of injury. Solithromycin is a new generation macrolide antibiotic.it has shown beneficial effect on hepatocyte ballooning and inflammation in murine models of diet induced NASH. ANTIFIBROTIC MEDICATIONS Simtuzumab is a monoclonal antibody against lysyl oxidase-like 2 (LOXL2), a matrix enzyme responsible for cross-linking of collagen chains that is expressed extensively in fibrotic regions in the liver. 18 Recommendations Relatively small amounts of weight loss reduce liver fat and improve hepatic IR. Structured programmes aimed at lifestyle changes towards healthy diet and habitual physical activities are advisable in NAFLD. Patients without NASH or fibrosis should only receive counselling for healthy diet and physical activity and no pharmacotherapy for their liver condition. In overweight/obese NAFLD, a 7 10% weight loss is the target of most lifestyle interventions, and results in improvement of liver enzymes and histology. Drug therapy should be indicated for progressive NASH (bridging fibrosis and cirrhosis) but also for early-stage NASH with increased risk of fibrosis progression (age >50 years; diabetes, MetS, increased ALT) or active NASH with high necroinflammatory activity. There is scarce evidence for a histological efficacy of metformin in NASH. 19 While no firm recommendations can be made, pioglitazone (most efficacy data, but off-label outside T2DM) or vitamin E (better safety and tolerability in the short-term) or their combination could be used for NASH. The optimal duration of therapy is unknown. In patients with increased ALT at baseline, treatment should be stopped if there is no reduction in aminotransferases after 6 months of therapy while in patients with normal ALT at baseline, no recommendations can be made. Statins may be confidently used to reduce LDL cholesterol and prevent cardiovascular risk, with no benefits or harm on liver disease.

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11 19. Shields WW, Thompson KE, Grice GA, Harrison SA, Coyle WJ. The effect of metformin and standard therapy versus standard therapy alone in nondiabetic patients with insulin resistance and nonalcoholic steatohepatitis (NASH): a pilot trial. Therap Adv Gastroenterol 2009;2: