Accepted Manuscript

Perspectives on Treatment for Nonalcoholic Steatohepatitis

Guillaume Lassailly, Robert Caiazzo, Franois Pattou, Philippe Mathurin

PII:
DOI:
Reference:

S0016-5085(16)00323-1
10.1053/j.gastro.2016.03.004
YGAST 60356

To appear in: Gastroenterology

Accepted Date: 8 March 2016
Please cite this article as: Lassailly G, Caiazzo R, Pattou F, Mathurin P, Perspectives on Treatment for
Nonalcoholic Steatohepatitis, Gastroenterology (2016), doi: 10.1053/j.gastro.2016.03.004.
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Perspectives on Treatment for Nonalcoholic Steatohepatitis

Guillaume Lassailly1,2,3, Robert Caiazzo4,5,6, Franois Pattou4,5,6, and Philippe Mathurin1,2,3*.

59000 Lille, France

2. Inserm, U995, F-59000 Lille, France

4. Univ. Lille, U1190, EGID, F-59000 Lille, France

5. Inserm, U1190, F-59000 Lille, France

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6. CHRU Lille, Service de chirurgie endocrinienne

*Corresponding author:
Prof. Philippe Mathurin
2 rue Michel Polonovski
59037, Lille CEDEX

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France
Phone : 03 20 44 53 21

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3. CHRU Lille, Service des Maladies de lappareil digestif

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1. Univ. Lille, U995 - LIRIC - Lille Inflammation Research International Center, F-

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Email : philippe.mathurin@chru-lille.fr

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Abstract
It is important to provide treatment to patients with nonalcoholic steatohepatitis (NASH)
because one third of patients with the metabolic syndrome die from liver disease. Basic
research studies have elucidated mechanisms of NASH pathogenesis, which could lead to

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therapeutic targets. Health agencies have confirmed strategies for the optimal management of
NASH and approved new drugs and treatments, which are urgently needed. The United States
Food and Drug Administration recently endorsed endpoints for NASH therapy. The reversal
of NASH with no evidence of progression to advanced fibrosis has been defined as the

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endpoint for phase 2b and phase 3 trials in patients with NASH and early-stage fibrosis.
Although a decrease in the non-alcoholic fatty liver disease activity score could serve as an
endpoint in clinical trials, it is not clear whether patients with lower scores have a lower risk

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of progression to advanced fibrosis. Endpoints for clinical trials of patients with NASH
cirrhosis are currently based on model for end-stage liver disease and Child-Pugh-Turcotte
scores, as well as the hepatic venous pressure gradient. Different strategies are being explored
to reduce liver diseases that are linked to a sedentary lifestyle, overeating, and genetic factors.
In association with insulin resistance and deregulation of the lipid metabolism (accumulation

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of lipotoxins that promote hepatic lipogenesis, adipose tissue lipolysis, and impaired oxidation), these factors could increase the risk of liver steatosis with necroinflammatory
lesions and fibrosis. We review the pathogenic mechanisms of NASH and therapeutic options,
as well as strategies that are being developed for treatment of injury to the liver and other

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organs.

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The optimal treatment for non-alcoholic steatohepatitis (NASH) would reduce liver-related
mortality, metabolic comorbidities, and the risk of cardiovascular events. Research aimed at
achieving these goals has received a large amount of support because NASH has been
declared a public health issue by public health authorities. The endorsement of therapeutic

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endpoints for NASH by the United States (US) Food and Drug Administration (FDA) was
important and facilitates development of new agents. The reversal or resolution of NASH,
defined as the disappearance of necroinflammatory features (hepatocyte ballooning and portal
inflammation) in histologic analysis, along with an absence of evidence for disease
progression to advanced fibrosis, has been recognized as the endpoint for phase 2b and phase

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3 trials of patients with NASH and early-stage fibrosis1. Although a decrease in the nonalcoholic fatty liver disease (NAFLD) activity score (NAS) could be used as an endpoint in

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clinical trials, studies are needed to determine whether patients with lower scores have a
reduced risk for progression to advanced fibrosis. Endpoints for clinical trials of patients with
NASH and cirrhosis are currently based on model for end-stage liver disease and Child-PughTurcotte scores, as well as the hepatic venous pressure gradient1. These new approaches
provide a foundation to develop treatments for NAFLD.

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New therapeutic targets must be identified that can lead to development of agents to
reduce disease progression in patients with NASH. To limit liver-related complications, these
agents should target 1 of the several pathways in the complex process of liver injury in
patients with NASH. These are likely to involve agents that modify the metabolic profile,

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because accumulation of hepatic fat and liver injury are associated with insulin resistance2.
We review the roles of weight loss, insulin sensitization, lipid metabolism, oxidative stress,

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fibrosis, inflammation and the intestinal microbiota in development and treatment of NASH.

Weight Loss

Diet and physical activity

Patients with NAFLD have a higher daily caloric intake and are more sedentary3 than those
without. The first step in the management of NASH is to obtain significant and sustained
weight loss by changing the patients lifestyle through diet and physical activity. Weight loss
increases hepatic and peripheral insulin sensitivity, reduces oxidative stress, and improves the
lipid profile. A restricted calorie diet rapidly decreases steatosis, improves insulin sensitivity,
and optimizes endogenous glucose synthesis4.

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Hepatic triglyceride content decreases with a weight loss of 3%5%, but further
weight loss is necessary to reduce necroinflammation. Peripheral (skeletal muscle) insulin
resistance improves with more than 7%4 of weight loss. In randomized trials with consecutive
biopsies, a decrease in the necroinflammatory process was mainly observed in the subgroup
that lost at least 7%9% of body weight.

5, 6

However, this did not affect the progression of

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fibrosis. A prospective study of 293 patients with NASH showed that 90% of steatosis
disappeared and fibrosis regressed in 45% of patients with a weight reduction >10%7. Weight
loss of >7%9% should therefore be the goal to reduce necroinflammation, whereas weigh
loss >10% should be the goal to induce regression of fibrosis in patients with NASH.

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On the other hand, further studies are necessary before specific diets can be
recommended for patients with NASH. Low-carbohydrate and Mediterranean diets seem to be
effective alternatives to low-fat diets, with different metabolic effects. Low-carbohydrate diets

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have a more positive effect on lipids whereas the Mediterranean diet improves control of
glycemia8. The daily maximum amount of fructose or sucrose has not been confirmed,
although there is increasing evidence for their negative effects (especially high-fructose corn
syrup), including increased visceral adipose tissue, liver fat accumulation, and insulin
resistance. A pilot study in 15 patients indicated that diets can reduce histologic markers of

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NASH. The prescribed diet included 40%45% carbohydrates (especially complex

carbohydrates with fiber), 30%40% fat (especially mono- and polyunsaturated fatty acids),
and 15%20% protein9. The Mediterranean diet is rich in mono-unsaturated fatty acid and
was more effective than a low-fat, high-carbohydrate diet in increasing insulin sensitivity in a

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randomized cross-over study of patients with NAFLD. However, this study did not perform
histologic analyses10. A study is underway to evaluate the effects of the Mediterranean diet in
patients with NASH (NCT01894438).

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Increased coffee consumption has been inversely associated with the risk of cirrhosis

or progression of fibrosis in patients at risk of liver disease, including NAFLD11-13. However

biases might have led to overestimates of coffee intake and risk of fibrosis progression.
Moreover, differences in coffee intake could reflect differences in socioeconomic status, or
exposure to other variables could affect fibrosis progression. Little is known about the
mechanisms by which coffee protects the liver, although a study an animal model of NASH
found that coffee reduced the amount of fat in the liver, oxidative stress, and inflammation.
Although coffee consumption is not associated with increased cardiovascular mortality,14 not
enough data are available to recommend increased coffee intake for patients with NASH.

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Besides diet, lifestyle change includes physical activity. The American Heart
Association recommends aerobic resistance activities, including at least 150 minutes
(preferably 300 minutes) of moderate-intensity physical activity per week, or at least 75
minutes

(preferable

150

minutes)

of

intense

cardiorespiratory

activity.

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recommendations may be applied to patients with NASH, although they have not been

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specifically evaluated in this subgroup. A recent study showed that a weekly combination of
250 min of moderate to vigorous exercise was more effective than 150 min in reducing
steatosis and liver stiffness. Like diet, physical activity must be based on realistic and
sustainable objectives15. The major difficulty is obtaining long-term complianceespecially

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in individuals who are not accustomed to regular intense exercise.

In conclusion, lifestyle therapy is insufficient to induce long-term weight loss and

resolve NASH. Only 10% of patients who commit to a lifestyle intervention lose more than

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10% of their weight, even when innovative approaches, such as brief consults with coaches,
are provided to instruct patients and about behaviors that control weight provide structured
recommendations16. These limits must be overcome for this strategy to be effective. A
therapeutic algorithm is needed to define 10% weight loss as the minimum goal for patients
with NASH and to offer alternative therapy to those who do not reach the goal or in whom

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NASH persists despite successful weight loss.

Weight loss with pharmacologic agents

Weight loss medications induce a median loss of approximately 3%9%17 A randomized

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study of patients in primary care showed that a combination of lifestyle intervention and a
prescription of weight loss medication (orlistat or sibutramine) produced a 10% loss of
baseline weight in a significantly higher proportion of patients (17.8%) than of those

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receiving brief lifestyle counseling alone (9.9%) or usual (6.2%) 16.

However, health agencies have discontinued or raised concerns about these

medications. Sibutramine, a serotonergic and noradrenergic reuptake inhibitor that promotes

satiety, was associated with a 16% increase in risk of serious cardiovascular events18 and was
taken off the market by the FDA. The European Medicine Agency (EMA) recommends
listing the risk for liver-related side effects, including severe liver injury and rare cases of
acute liver failure leading to death or liver transplantation, for orlistatan inhibitor of gastric
and pancreatic lipase. Nevertheless, the benefits of these drugs are considered to outweigh
their risks by health agencies. Orlistat did not modify necroinflammation or fibrosis in
patients with NASH 5, 19.

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Rimonabant20, a selective antagonist of central and peripheral CB1 receptors,
promotes satiety, reduces weight and waist circumference, and improves the lipid profile
through by increasing high-density lipoprotein (HDL) and reducing triglycerides21, 22. There
was great hope for this drug because targeting CB1 receptors in the liver reduces steatosis,
portal pressure, and the pathways of fibrogenesis23. These results were the rationale to test

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rimonabant in patients with NASH (NCT00577148; NCT00576667). Unfortunately the trial

was stopped due to serious psychiatric side effectspatients developed severe depression and
became suicidal21. Research is underway to develop a peripherally restricted CB1 antagonist
that reduces liver injury without causing neuropsychiatric events 23.

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Liragutide, an agonist of glucagon-like petide-1 (GLP1) is an anorectic incretin

hormone. It was found to be significantly more effective than orlistat in treating obese patients
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and increasing weight loss in patients with type 2 diabetes25. In addition to the benefits to

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body weight and glycemic control, GLP1 pathways seem to interfere with other mechanisms
of liver injury. Because of this profile, this drug is being investigated for treatment of
NASH26.

Bariatric surgery

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In severely obese patients (those with a body mass index [BMI] 40 or 3540 with
comorbidities), bariatric surgery induces sustained weight loss and has been recommended by
the National Institutes of Health for motivated candidates. Perioperative mortality varies from
0.1% to 0.3%, depending on the type of surgery and patient characteristics27, 28. Regardless of

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procedure type, bariatric surgery induces long-term weight loss (of 15%25%), as well as
remission of diabetes29-31 and reduced overall long-term mortality32particularly from
diabetes33, heart disease,34 and cancers35.

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The effects of bariatric surgery on the liver include reductions in steatosismainly

within the first year after surgery until 5 years later. The kinetics of the insulin resistance
profile parallel those of steatosis and ballooning, with the greatest reductions the first year
after surgery but continuing for up to 5 years. The long-term outcome can be predicted by
early improvement in insulin resistance following the procedure36. Nevertheless, reduced
insulin resistance is often not enough to resolve NASH. At the same time, some patients with
improvement in NASH injuries remain insulin resistant. Moreover, despite the ability of
bariatric surgery to reduce steatosis, there is controversy over its effects on fibrosis and the
necroinflammation. Prospective studies are needed to resolve this issue.

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Small, preliminary studies reported that NASH disappeared from approximately 85%
90% of patients who underwent gastric banding or bypass surgeries37, 38. A recent prospective
study that analyzed sequential liver biopsies from 1540 patients who underwent bariatric
surgery for morbid obesity provided strong evidence that the procedure resulted in the
disappearance of NASH. One hundred and nine patients had biopsy-proven NASH at

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baseline; NASH had disappeared from the livers of approximately 85% of the patients 1 year
later. NASH resolved a greater proportion of patients with mild disease than from those with
moderate or severe disease at baseline. Bariatric surgery significantly reduced all the
histological components of NASH including fibrosis that was improved in about 30% of cases
.

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39

After bariatric surgery, the extent of weight loss appears to associate with the
reduction in liver injury. Gastric bypass seems to be more effective than gastric banding in

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reducing liver injurythis specific benefit is believed to be due to the greater loss in weight40.
However, larger studies of patients matched for disease severity and comorbidities are needed
to confirm these results. Beside the ability of gastric bypass to promote weight loss, it has
several other features that make it more effective than gastric banding. For example, more
rapid contact of nutrients with the ileum increases satiety by increasing secretion of anorectic

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hormones such as PYY or incretins (GLP1 and GLP2). GLP1 is involved in the enteroinsular
axis, stimulating insulin secretion and decreasing hepatic glucose output and insulin resistance
in the liver and adipose tissues41.

So, bariatric surgery appears to reduce fibrosis and necroinflammatory processes to

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alter disease progression and prevent development of cirrhosis and its complications.
Cirrhosis is considered to be a contraindication to bariatric surgerymortality increased 21fold in patients with decompensated cirrhosis and 2-fold in patients with compensated

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cirrhosis42. However, it is not clear whether this contraindication applies to patients with
cirrhosis or portal hypertension or liver dysfunction. Another important area for future
research is to determine whether bariatric surgery is effective for severely obese patients who
are candidates for liver transplantation43, 44. A preliminary study of 7 patients reported that
sleeve gastrectomy performed at the same time than liver transplantation was more effective
to reduce weight of obese patients with end-stage liver disease that medical management.
Thus bariatric surgery could be of interest for the management of liver transplantation
candidates with severe obesity45.
In conclusion, although bariatric surgery resolves NASH in patients in whom lifestyle
therapy has failed, perioperative risks limit its application. Because of the high rate of

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remission of NASH, bariatric surgery could be tested in randomized controlled trials of
moderately obese patients with extensive fibrosis and severe comorbidities.

Insulin Sensitization
Insulin-resistance is an important factor in the development of NASH. Excess fatty-acid

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transport into hepatocytes is required for development of hepatic insulin resistance and
NAFLD (Figure 1). In rats fed high-fat diets, increased hepatic diacylglycerol content and the
translocation of protein kinase C epsilon (PRKCE) to the plasma membrane contributes to
development of insulin resistance. By binding the insulin receptor an inhibiting its activity,
translocated PRKCE2,

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impairs the ability of insulin to activate glycogen synthesis and

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inhibit gluconeogenesis . These mechanisms have been also been observed in liver tissues
from obese patients with NAFLD; in these patients, hepatic diacylglycerol content,

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cytoplasmic lipid droplets, and PRKCE activation correlated with reduced insulin signaling47.
Accumulation of fat in the liver therefore seems to be the first step in the development of
hepatic insulin resistance.

Studies from animal models indicate that liver inflammation is not sufficient for
development of hepatic insulin resistance, regardless of the pathway 48. The severity of insulin
resistance was associated with more severe necroinflammation injury in liver tissues from

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patients with NASH than those without NASH36. By increasing peripheral lipolysis, insulin
resistance increases delivery of free fatty acids to the liver,49 leading to excess fat and
increased -oxidation, which worsens oxidative stress. Insulin resistance might be targeted

Metformin

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therapeutically, perhaps with insulin sensitizing agents.

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By reducing gluconeogenesis50 metformin, a biguanide agent, increases fasting levels of

glucose, as well as levels of postprandial glucose and glycosylated hemoglobin; it is
recommended for obese patients with type 2 diabetes51. Metformin also reduces fat body mass
without changing lean body mass50. In a proof of concept study of 20 patients with NASH,
metformin was associated with decreased levels of aminotransferases and insulin resistance 52.
These effects were confirmed in a randomized controlled trial and a non-controlled
prospective study. Histologic features of NASH were reduced in 30% of liver tissues
analyzed; a higher proportion of patients from who these tissues were obtained lost weight
during metformin treatment53, 54. Nevertheless, other studies have not confirmed the ability of
metformin to reduce histologic features of NASH, and a meta-analysis concluded that

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metformin was not effective in patients with NASH55. The 2012 American Association for the
Study of Liver Disease (AASLD) guidelines do not recommend the use of metformin in
adults with NASH56.
More recent results have indicated that metformin could decrease the risk of
hepatocellular carcinoma, in a dose-dependent manner, in obese patients with type 2
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. Metformin might therefore be used to decrease the risk of liver cancer in

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diabetes57,

patients with diabetes, but this interesting strategy requires further study58.

Peroxisome proliferator-activated receptors (PPARs)

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Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors involved in lipid

and glucose homeostasis and regulation of inflammation and cellular differentiation.
Randomized studies of patients with type 2 diabetes have shown that PPAR agonists

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decrease glucose and glycosylated hemoglobin. Because PPAR agonists such as

rosiglitazone and pioglitazone have multiple targets, they have been extensively tested in
patients with NASH. In a randomized study, patients with NASH given rosiglitazone had
significant reductions in levels of aminotransferases, insulin resistance, and steatosis, but no
changes in necroinflammation or fibrosis, compared with placebo group. However, there was
no additional benefit in extending rosiglitazone therapy up to 2 years.

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In 2 randomized trials of pioglitazone,59, 60 patients with impaired glucose tolerance or

type 2 diabetes had significant reductions in levels of aminotransferases, steatosis (by 65%),
hepatocyte ballooning, necrosis, and lobular inflammation, along with increased hepatic

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insulin sensitivity, compared to patients given placebo. However, a significant reduction in

fibrosis was only reported in only 1 study60. A large randomized controlled trial of in nondiabetic patients with NASH61 compared pioglitazone to placebo, as well as vitamin E to

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placebo. Although the pioglitazone group did not reach the pre-specified statistical
significance for the primary outcome (that was a reduction of 2 point in NAS) compared to
the placebo group, patients given pioglitazone had significant reductions in steatosis,
inflammation, hepatocyte ballooning, insulin resistance, and levels of liver enzymes61.
Pioglitazone seemed to be more effective than vitamin E in producing the secondary
outcomeresolution of NASH, although there was no statistical analysis performed in the
comparison of these agents61.
Because of the promising results from these trials, pioglitazone is listed as a potential
treatment for NASH in the 2012 AASLD guidelines56. However, there is concern about the
safety of PPAR agonists. All large randomized controlled trials of pioglitazone and

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rosiglitzone have shown weight gain of 35 kg after patients took these drugs59,

61, 62

Furthermore, the FDA and EMA have warned about the cardiovascular risk related to the use
of PPAR agonists63, 64. Rosiglitazone has been associated with an increased risk of cardiac
ischemia and heart failure, and its prescription was restricted by the FDA. Data that indicate
that pioglitazone might induce congestive heart failure, although no increased risk of cardiac

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death was found among the pioglitazone group. Expert guidelines have therefore approved the
prescription of pioglitazone for patients with NASH with safety and caution recommendations
on long-term use56.

Elafibranor (GFT505)65 is an agonist of PPAR and PPAR that has been tested in

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patients with NASH because it increases hepatic insulin sensitivity, improves glucose
homeostasis and lipid metabolism, and reduces inflammation. In animal models of NAFLD or
NASH and liver fibrosis, elafibranor reduced steatosis, inflammation, and fibrosis as well as
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. In clinical trials, elafibranor (NCT01271751,

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markers of inflammation and fibrogenesis

NCT01275469, NCT01275469, and NCT01271777) significantly improved patients

metabolic profiles and reduced levels of aminotransferases. A recent randomized controlled
phase 2 study of 274 patients with NASH compared 2 doses of elafibranor to placebo. There
was no significant difference between the number of patients in each group who reached the

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primary endpoint (resolution of NASH without the worsening of fibrosis). However, after
correction for baseline severity and the center effect, the highest dose of elafibranor (120 mg
per day) 64 resolved NASH in significantly more patients than placebo. These results must be
confirmed in a well-designed phase 3 study of patients with severe NASH and fibrosis, which

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seems to be the population who can receive the most benefit from this agent.

The GLP1 pathway

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GLP1 is an incretin hormone that is secreted after nutrients come in contact with the ileum. It
increases insulin secretion, decreases hepatic glucose output and insulin resistance in the liver
and adipose tissues, and promotes satiety. The GLP1 pathway decreases liver fatty acid
accumulation through the activation of numerous genes such as PPAR/ witch will enhance
hepatic fatty oxidation, lipid export, and insulin sensitivity. This pathway also stimulates
macro- and chaperon-mediated autophagy, and to reduce endoplasmic reticulum stress and
TNF, IL6, IL1, and MCP1/CCL2 expression4, lipid export, and insulin sensitivity. This
pathway also stimulates macro- and chaperon-mediated autophagy and reduces endoplasmic
reticulum stress and expression of tumor necrosis factor (TNF), interleukin-6 (IL6), IL1B, and

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MCP1 (also called CCL2)4. All these GLP1-related mechanisms decrease liver inflammation
and apoptotic liver injuries66.
As previously observed in patients with diabetes and obese patients24, 25, liraglutide
reduces weight loss in patients with NAFLD, but also decreases levels of fasting glucose and
HbA1c, insulin resistance, and peripheral lipolysis, leading to lower levels of low-density

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lipoprotein (LDL)cholesterol, serum leptin, and adiponectin. The most frequent side effects
are gastrointestinal symptoms, which occur in approximately 20% of cases. Levels of
aminotransferases, gamma-glutamyl transferase, and steatosis also improved67. Researchers
performed a phase 2 randomized controlled trial (LEAN study)26 of 52 patients with NASH

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and type 2 diabetes; 46% of patients had Kleiner fibrosis stage F3 and 58% had fibrosis stage
F4. The authors observed NASH resolution in a significantly higher proportion of patients
who received liraglutide (39% of patients) than those given placebo (9% of patients), along

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with reductions in fasting level of glucose and significantly less worsening of fibrosis in
patients who received liraglutide. In a post-hoc analysis, there was no difference in weight or
glycemic control in the liraglutide group between patients in whom NASH resolved vs those
without NASH resolution. Moreover, liraglutide reduced biomarkers of fibrosis, indicating
that in addition to studies of weight loss, larger studies should be performed to evaluate the

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ability of this agent to reduce fibrosis. Large phase 3 randomized controlled studies are also
needed to determine the effects of liraglutide on NASH, considering there was no significant
difference in level of aminotransferase levels between groups, as well as the low spontaneous
resolution (9%) of NASH in patients who received the placebo. Exenatide, another agonist of

available.

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GLP1, is being evaluated in a trial of patients with NASH (NCT01208649), but no data are

An alternative approach is to maintain activation of the GLP1 pathway. The incretin

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inhibitor dipeptidyl peptidase 4 (DDP4), which is involved in the degradation of GLP1 and
induces a sustained metabolic effect, has been tested in patients with type 2 diabetes. Health
agencies have approved saxagliptin and sitagliptin for treatment of patients with type 2
diabetes, but cardiovascular safety is a subject of debate. Randomized clinical trials have
evaluated the vascular effects of saxaglitin in patients with type 2 diabetes and found an
acceptable level of cardiovascular risk, with no increase in mortality, although the rate of
hospitalization for heart failure increased among patients receiving this agent68. There was no
increased risk of cardiovascular events or heart failure with sitagliptine69. Steatosis decreased
in animal models following treatment with sitagliptine70. Researchers are considering a
clinical trial of sitagliptine in patients with NASH (NCT01963845).

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Obeticholic acid
Obeticholic acid, a 6a-ethyl derivative of chenodeoxycholic acid, is an active ligand of
nuclear receptor subfamily 1 group H member 4 (NR1H4 or FXR), which regulates glucose,
lipid, and energy homeostasis71, 72 and acts in anti-inflammatory and anti-fibrosis pathways73,
. In patients with type 2 diabetes and NAFLD, 25 and 50 mg of obeticholic acid per day

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74

induced weight loss and decreased insulin resistance and serum markers of liver inflammation
and fibrosis75. In an interim analysis of results from a phase 2b randomized controlled trial of
283 patients with NASH, 25 mg obeticholic acid was more effective than placebo at

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producing a decrease in NAS of 2 points or more (the primary endpoint), with no worsening
of fibrosis. In fact, fibrosis was significantly reduced in patients who received obeticholic
acid, compared to patients given placebo; these patients also had reductions in steatosis,

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hepatocellular ballooning, and lobular inflammation76.

However, concerns were raised about the observation that patients who received
obeticholic acid had higher concentrations of total serum cholesterol and LDLcholesterol,
but lower levels of HDLcholesterol. This is a major issue due because these factors increase
the risk of cardiovascular events in this population. Therapeutic strategies that include this

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agent might need to include a statin, to limit hyperlipemia. Another limitation of obeticholic
acid is that about 20% of patients who take it develop pruritus, which could reduce
compliance.

The optimal dose of obeticholic acid was evaluated in a phase 2 dose-range study of

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200 patients with NASH that used a decrease in NAS of 2 points or more as the primary
endpoint. . A statistically significant difference was observed only in group that received 40
mg obeticholic acid, compared to placebonot in the groups that received 10 or 20 mg
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. The long-term safety and efficacy of the 25 mg/day dose of obeticholic

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obeticholic acid

acid will be evaluated in an international phase 3 randomized controlled trial

(NCT02548351). This trial will focus on reduction in liver-related morbidity and mortality, as
well as cardiovascular events.

Altering Lipid Metabolism

Polyunsaturated fatty acids (PUFAs) can either promote or inhibit inflammation, depending
on their structure78. N6 PUFAs, such as linoleic acid and arachidonic acid have been shown
to promote inflammation and synthesis of eicosanoids79. Conversely, n3 PUFAs and omega3

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fatty acids, such as linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid
reduce inflammation. In animal models, a diet enriched in n3 PUFAs and omega3 fatty acids
reduced steatosis by inhibiting sterol regulatory element binding protein 1c (SREBF1), which
regulates expression of genes involved in glucose and lipid metabolism80.
The ratio of n-6:n-3 is altered in serum samples from patients with NAFLD81. A

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randomized placebo-controlled trial of 243 patients with NASH or severe NAFLD (NAS
scores of 4 or more) showed that 1.8g2.4g/day of ethyleicosapentanoic acid (EPA-E), a
synthetic n-3 PUFA, had no significant effect on liver enzymes, insulin resistance, steatosis,
or histologic features of NASH82. Primary analysis of data from a randomized controlled trial

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of 103 patients with NAFLD did not find any significant difference between the combination
of docosahexaenoic acid plus EPA vs placebo reducing amounts of liver fat83.
Inhibition of stearoyl-CoA desaturase (SCD) activity by aramchol (arachidyl amido

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cholanoic acid) lowers the amount of fat in the liver by decreasing synthesis and increasing
oxidation84. In addition to the reduction of fatty acid synthesis, the SD1 deficiency seems to
be associated with an increased activity of the carnitine palmitoyltransferase system, which
leads to long chain fatty acid oxidation85. In a randomized controlled trial of 60 patients
with NAFLD, 300 mg/day aramchol decreased the amount of fat in the liver but did not

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significantly affect liver enzymes86.

Leptin is an adipokine that inhibits food intake and decreases hepatic glucose
production and insulin resistance. Furthermore, leptin decreases steatosis, by reducing de
novo synthesis of fatty acids by inhibiting SCD, and increasing fatty acid -oxidation87.

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Administration of a recombinant leptin (metreleptin) reduced fatty liver in patients with

general lipodystrophy, a genetic disorder that causes leptin deficiency88, 89. Data from longterm studies have shown that although metreleptin significantly reduced steatosis, lobular

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inflammation, and hepatocellular ballooning, it did not reduce fibrosis89. Resistance to leptin
contributes to the pathogenesis of obesity and could account for the correlation between the
amount of body fat and serum level of leptin in obese patients87. However, the best
therapeutic strategy for modulation of the leptin pathway is uncertain, because of the different
effects of leptin on metabolism, the inflammatory processes, and fibrogenesis.
Combination treatment strategies that include a lipid-lowering agent have produced
some promising results. For example, the NiemannPick C1like 1 (NPC1L1) protein
mediates

intestinal

absorption

of

cholesterol

whereas

the

proprotein

convertase

subtilisin/kexin type 9 (PCSK9) (inhibited by alirocumab and evolocumab) is involved in the

regulation of cholesterol homeostasis through modulation of the LDL receptor in hepatocytes.

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The combination of ezetmibe (a NPC1L1 inhibitor), or PCSK9 inhibitor (alirocumab or
evolocumab) with statins decreases levels of LDLcholesterol and improves cardiovascular
outcomes by reducing the risk of myocardial infarction and ischemic stroke90-94. PCSK9knockout mice fed with high-fat diet are protected for the development of steatosis, compared
with controls95. Mice given ezetimibe, which inhibits NPC1L1, have reduced inflammation

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and fibrosis biomarkers96. A randomized controlled trial of 50 patients with NASH (the
MOZART trial)97 compared the effects of ezetimibe with placebo and significant difference in
histologic markers of response, serum levels of aminotransferases, or changes in liver
stiffness (assessed by magnetic resonance elastography). Further studies are needed to

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determine the effects of PCSK9 inhibitors.

Reducing Oxidative Stress

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Oxidative stress causes chronic tissue injury, leading to increased cell death and fibrogenesis.
There are multiple sources of oxidative stress involved in NAFLD (Figure 1). Immune cells
such as peripheral mononuclear cells and macrophages produce excessive reactive oxygen
and nitrogen species under conditions of chronic liver inflammation. Reactive oxygen species
can disturb the intracellular redox system. Intracellular redox balance can also be disrupted by
metabolic abnormalities such as lipid oxidation or endoplasmic reticulum stress, leading to

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mitochondrial injury and cell death98. Numerous anti-oxidant agents have therefore been
tested in patients with liver disease. Except for Vitamin E, the effects of these agents in

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patients with NASH have been disappointing.

Vitamin E or tocopherol

The vitamin E isoform rrr-alpha-tocopherol has been shown to have greater efficacy than the

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other 7 isoforms of vitamin E in patients with NASH. Vitamin E is considered to be the firstline pharmacotherapy for non-diabetic patients with NASH. However, further studies are
necessary before vitamin E can be recommended for diabetic patients with NASH or with
cirrhosis

56

. In a randomized controlled trial (the PIVENS trial) of 247 non-diabetic patients

with NASH, a daily dose of 800 mg vitamin E for 96 weeks reduced steatosis, lobular
inflammation, hepatocellular ballooning, and NAS, compared to placebo61. The safety profile
of vitamin E did not differ from that of placebo. Subjects receiving vitamin E had early and
continued decreases in levels of aminotransferases. Alkaline phosphatase and -glutamyl
transpeptidase levels progressed in a similar manner. However, after vitamin E was
discontinued, aminotransferase levels returned to the same range as that of placebo. This

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relapse after 96 weeks of vitamin E administration indicates that it might need to be given for
a longer time periods, or indefinitely, to patients with NASH, because in most cases, the
factors that contribute to liver injury persist.
This is an important consideration, because long-term vitamin E use was associated
with increased mortality99. Long-term use of vitamin E was associated with a 22% increase in

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risk of hemorrhagic stroke and a 10% reduction in thrombotic stroke. However the effect of
vitamin E on overall stroke events has not been clarified100. Concerns about the safety profile
associated with long-term use of vitamin E were raised in a randomized controlled trial (the
SELECT study) of 32,500 men that investigated whether selenium and/or vitamin E could

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prevent prostate cancer. After a preplanned 7-year interim analysis, the safety committee
recommended discontinuing the study because futility analysis did not find any evidence that
selenium or vitamin E affected risk for prostate cancer101. This result was confirmed by the

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Physicians' Health Study II, which followed 14,641 patients who were randomly assigned to
groups given placebo or vitamin E for 8 years and did not find any difference in the risk of
prostate cancer between groups102. However, the study committee of the SELECT trial
recommended that the preplanned final analysis be performed 7 years after the last patient
was randomly assigned to their group, because there was a trend toward an increase in risk of

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prostate cancer in the vitamin E group. The final analysis showed a 17% increase in the
incidence of prostate cancer among individuals with an average risk of prostate cancer who
received 400 mg/day of vitamin E103. However, no firm conclusions can be drawn because of
the contradictory conclusions of meta-analyses evaluating the risk of vitamin E-related

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mortality.

Non-vitamin E antioxidants

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Ursodeoxycholic acid does not significantly affect histologic features of NASH56,

104, 105

Antioxidants such as silymarin, cysteamine bitartrate, or resveratrol have been studied in

proof-of-concept studies, but there is no evidence to support their further evaluation in
patients with NASH 56.

Preventing or Reducing Fibrosis

Anti-fibrotic agents are being developed to prevent progression of NASH106. One strategy
targets factors that promote chronic liver inflammation, to prevent subsequent fibrogenesis
(such as antiviral agents)

107

. Reduced fibrosis is therefore an endpoint for studies of the

effectiveness of these agents in patients with NASH. Another approach targets the

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pathogenesis of fibrosis, based on clinical evidence that NASH-related mortality is associated
with the severity of fibrosis106 .
Simtuzumab (GS-6624) is an antibody against enzyme lysyl oxidase-like-2 (LOXL2),
which is overexpressed during development of liver fibrosis and promotes cross-linking of
fibrillar collagen I. In animal models, an anti-LOXL2 agent reduced liver and lung fibrosis by

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inhibiting fibroblasts and decreasing growth factors and the transforming growth factor-
pathway108. Patients with NASH and stage 3 and 4 fibrosis have been enrolled in a study of its
safety and efficacy (NCT00799578; NCT01672879). Data are not yet available.

Galectins are a family of proteins with binding specificities for -galactoside sugars.

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Galectin-3 has crosslinking and adhesive properties and is encoded by the lectin, galactosidebinding, soluble, 3 gene (LGALS3); it is a target of direct-acting anti-fibrotic agents. This
protein is mediates fibrogenesis in the liver and other organs. Lgals3/ mice are resistant to
109

. Phase 1 studies of galectin-3 inhibitors

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induction of liver fibrosis by toxin administration

have been completed (GR-MD-02, NCT01899859) (see Table 2) and the agent is being
evaluated in phase 2 studies of patients with NASH and cirrhosis (NCT02462967) or
advanced fibrosis (NCT02421094).

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Reducing Cell Death and Inflammation

Pentoxifylline appears to prevent inflammation-related liver injury by decreasing production

of TNF, which reduces insulin resistance, oxidative stress,

110

and cell death. Pilot studies

have reported beneficial effects of pentoxifylline, and a randomized controlled trial of 50

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patients with NASH found that pentoxifylline reduced steatosis, lobular inflammation, and
NAS, but had no significant effect on hepatocyte ballooning or fibrosis111. A meta-analysis
concluded that although pentoxifylline had a moderate effect in patients with NASH, there

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was not enough robust evidence to show that it was better than placebo112. Larger trials are
needed to evaluate the effectiveness of pentoxifylline.
Modulators of apoptotic signaling could reduce inflammation and liver cell injury in

patients with NASH. GS-9450, which has selective activity against caspases 1, 8, and 9, was
tested in a randomized controlled trial of 124 patients with NASH. Patients given GS-9450 for
4 weeks had reduced caspase-3cleaved cytokeratin-18 fragments in liver tissues and lower
levels of alanine aminotransferase than patients given placebo113. However, development of
this agent was discontinued because some patients developed liver injury. This should be
carefully evaluated because drug-induced liver injury has been associated with other caspase
inhibitors, indicating a possible class effect.

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Agents that target chemokine (C-C motif) receptor 2 (CCR2) or CCR5 might
be developed for patients with NASH, because the chemokines they bind can increase insulin
resistance and recruit macrophages to the liver and adipose tissue114. However, there is no
clear evidence for the role of CCR5 in the pathogenesis of NAFLD. This receptor regulates
CD8+ T-cell accumulation115 and the response to viral116 and bacterial infections117.

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Cenicriviroc, which inhibits CCR2 and CCR5, was initially developed for treatment of
patients with HIV infection. It will be evaluated in patients with NASH.

New Approaches

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There are several drugs in phase 1 development with new targets and previously
undescribed mechanisms of action (see Table 2). One class is the apical sodium-dependent
bile acid transporter (ASBT) inhibitor. ASBT is an ileal bile acid transporter that reabsorbs

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bile acids and seems to be required for enterohepatic recirculation and lipid homeostasis118.
Recent research into the pathways activated by bile acids (such as FXR agonists) found that
bile acid sequestrants have interesting metabolic effects, such as reducing blood glucose and
LDL in patients with type 2 diabetes119. Blocking bile acid absorption with ASBT inhibitors
could therefore reduce insulin sensitivity and potentially NAFLD.

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The mitogen-activated protein kinase kinase kinase 5 (also known as apoptosis signal
kinase-1 [ASK1]) is involved in oxidative stress-induced apoptosis

120

. Although data are

limited, ASK1 inhibitors could be of interest for treatment of NASH.

The composition of the intestinal microbiota has been associated with obesity, insulin
122

. NASH has been associated with

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resistance, and liver diseases including NAFLD121,

specific microbiota profiles, and inflammasome-mediated dysbiosis regulates progression of

NAFLD and obesity121,

122

. Transfer of intestinal microbiota from lean donors increased

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insulin sensitivity in individuals with metabolic syndrome

123

. Some studies have aimed to

alter the composition of microbiota in patients with NASH124. However, more studies are
needed to identify the microbes that affect insulin resistance, obesity, and liver cell injury125
and their mechanisms.

Future Directions
Although lifestyle intervention is still the first-line therapy for patients with NASH, only a
few patients are able to lose 10% of their body weight, which has been set as the threshold
required for NASH resolution. More efficient lifestyle and weight loss strategies are needed.
Bariatric surgery is highly effective in severely obese patients with NASH, and may be

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proposed for subjects who fulfill the validated criteria for this option. Expert guidelines
recommend vitamin E as the first-line therapy for non-diabetic patients with NASH.
However, many agents are in development for treatment of NASH, including obeticholic acid,
liraglutide, and elafibranor. Many studies are needed to determine the ability of these
molecules to resolve NASH and their long-term effects on liver-related mortality and

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morbidity.

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Table 1. Agents Available or in Development for Treatment of NASH or NAFLD
Drugs &

Action

Phase of

Therapy

Histological Impact

Pros & cons

Ref

Depends on the level of

Only 10% of patients reach the

6, 7, 56

10% weight loss threshold

development

Reduces insulin

Lifestyle

Recommended in

resistance,cardiovascular

first-line

weight loss:

risk; lipid profile reduces

treatment

>3%5%: reduces

oxidative stress

steatosis

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Weight loss

required for an effect, and risk

weight gain relapse

> 7%9%: reduces

necroinflammation

Induces sustained weight

Cohort studies

Reverses NASH in 85%

Recommended for patients

loss at least up to 10 years;

of patients. Reduces

with morbid obesity. Post-

increases insulin resistance

steatosis,

operative morbidity limits

and cardiovascular risk

necroinflammation, and

applicability to patients with

fibrosis

BMIs 35kg/m.

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Bariatric surgery

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10%: reduces fibrosis

37-39

Insulin sensitizers
Pioglitazone

PPAR agonist, lipid

homeostasis, inflammation,

GLP1 agonist, promotes

Pros: effective for patients

completed

steatosis

with NASH

necroinflammation

Cons: Weight gain

fibrosis

Risk of congestive heart

Phase 3

59, 61

failure (no effect on mortality)

and bladder cancer

Reduces

Pros: marketed for patients

satiation, weight loss,

steatosis

with diabetes, safety seems

reduces insulin resistance

necroinflammation

acceptable

No worsening of fibrosis

Cons: gastrointestinal side

resolution of NASH

effects, requires subcutaneous

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Liraglutide

Improves:

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and cell differentiation

Phase 2

26

administration

Agents with multiple targets

PPAR and PPAR

Reduces

120 mg per day reduces

Elfibranor

agonist;

steatosis

histologic features of NASH

reduces insulin resistance,

necroinfammation

inflammation, and fibrosis;

fibrosis

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GFT505/

Phase 3

improves lipid metabolism

Obeticholic acid

FXR agonist, increases

65, 126

Reverses NASH *
Phase 3

Reduces

Pros: reduces histologic

energy homeostasis and

steatosis

features in NASH.

lipid metabolism; reduces

necroinflammation

Cons: side effects, with 20%

insulin resistance,

fibrosis

of patients developing pruritus

inflammation, and fibrotic

NAS without worsening

pathways

of fibrosis

72,73

Agents that alter lipid metabolism

Aramchol

Inhibition of stearoyl
coenzyme A desaturase 1

Phase 2

Decrease fat amount, no

No effects on NASH, only

effects on NASH

preliminary results available

83

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(SD1)

Anti-oxidants
Vitamin E

Tocopherol, anti-oxydative
stress

Phase 2
completed

Significant effects on

Pros: recommended as first

steatosis

pharmacologic therapy

necroinflammation

Cons: no data from patients

NAS

with diabetes or cirrhosis;

61

concerns for long-term use

Pentoxifylline

Reduces inflammation
(potentially by inhibiting

Phase 2
completed

Reduces

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Agents that reduce inflammation or cell death

Pros: pilot studies indicate

steatosis

efficacy

TNF), blood viscosity, and

necroinflammation

platelet aggregation

NAS

Cons: lack of large

randomized controlled trials or

robust data sets

Inhibit CCR2 and

Phase 2

CCR5, reduces

Initially developed for

treatment of HIV infection.

insulin resistance and

Potentially interesting effects

in patients with NASH but

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recruitment of
inflammatory cells

No data

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Cenicriviroc

further studies are needed

*Did not achieve primary endpoint in large Phase 2 randomized controlled trial (resolution of NASH without worsening of
fibrosis). Statistical significance was obtained from subgroup analysis. Findings must be confirmed in phase 3 randomized

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controlled trials.

110

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Table 2. Agents in Development
Target

Potential Mechanism of

ClinicalTrial.gov

Action

identifier

Galectin-3

Anti-fibrotic

NCT01899859

PX-102

FXR agonist

Reduces insulin resistance

NCT01998659

SHP-626

ASBT inhibitor

Increases levels of GLP1

NCT02287779

GS-4997

ASK1 inhibitor

Reduces consequences of

NCT02466516

GR-MD-02

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Drug

Toll-like receptor 4

Anti-inflammatory agent

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JKB-121

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oxidative stress

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Note: All agents are either in phase 1 trials or pre-clinical studies

NCT02442687

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Figure 1. Pathophysiology of NASH and Therapeutic Targets

Legend:

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Patients with NASH have a higher daily caloric intake (especially high fructose corn syrup)
and are more sedentary than those without. Those bad habits are also associated with obesity
and metabolic syndrome witch are highly prevalent in NASH. Thus, lifestyle therapy with diet
and increased physical activity to obtain 10% of weight loss is the first step for NASH

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treatment. For a better efficacy, complementary weight loss strategies can be proposed as
bariatric surgery for severely obese patients. In NASH there is an excessive transport of sugar
and fatty-acids (from diet and increased lipogenesis) to the hepatocyte that leads to steatosis.

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Fat absorption may be targeted to reduce steatosis, as for example NCP1L1 (NiemannPick
C1like 1 protein) inhibitor (ezetimibe) that decreases the gut absorption of cholesterol.
However, steatosis is a key event for the development of hepatic insulin resistance. At a
molecular level, accumulation of fatty-acids in the hepatocytes enhances the translocation to
membrane of the primary novel PKC isoform epsilon that inhibits the insulin receptor

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activity. Hepatic insulin resistance increases lipogenesis and reduces fatty acids -oxidation
that lead to steatosis. This last mechanism can by reduced with aramchol, an inhibitor of
stearoyl coenzyme A desaturase (SCD) activity that promotes liver fat wash out through
increased -oxidation and reduced lipogenesis. In the therapeutic landscape for NASH, many

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agents target insulin resistance (liraglutide, PPAR agonist, metformin) that is a main
pathway of the pathogenesis of NASH. Insulin resistance can almost be consider as a
prerequisite target for the action of multiple hit drugs as obeticholic acid and elafibranor. But,

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insulin resistance is not sufficient alone to induce NASH. Therefore macrovacuolar lipid
droplets accumulation in the hepatocyte drives major cellular stress and injuries through 3
main pathways: mitochondrial injury, oxidative and endoplasmic reticulum stress. Oxidative
stress reduces phosphorylation of FOXO1, a nuclear factor involved in glucose production
and adipose storage. The non-phosphorylate FOXO1 is translocated to the nucleus and
increases hepatic glucose production that occurs in insulin resistance and diabetes.
Furthermore the activation of the MAP kinase pathway through p38 promotes inflammatory
pathways and increases apoptosis. Thus, inflammation and apoptosis are two therapeutic
targets for NASH. For anti-inflammatory treatments, anti-oxidant therapies as vitamin E
reduces inflammatory injuries due to oxidative stress. Dual CCR2 and CCR5 antagonist

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(Cenicriviroc) reduces the recruitment of inflammatory cells. The apoptotic pathway may be
targeted with anti-caspase therapy. Decreasing inflammation and apoptosis might reduce
fibrogenesis. Finally, fibrosis can be considered as the last target for therapeutic agents, as an

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example, anti-LOXL2 agents. (FFA:free fatty-acids, FA :fatty-acids, PKC: protein kinase C)

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