2016 Perspectives on Treatment for Nonalcoholic Steatohepatitis. GASTROENT

8/18/2019 2016 Perspectives on Treatment for Nonalcoholic Steatohepatitis. GASTROENT

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Accepted Manuscript

Perspectives on Treatment for Nonalcoholic Steatohepatitis

Guillaume Lassailly, Robert Caiazzo, François Pattou, Philippe Mathurin

PII: S0016-5085(16)00323-1

DOI: 10.1053/j.gastro.2016.03.004

Reference: 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

, François Pattou4,5,6

, and Philippe Mathurin1,2,3*

.

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

59000 Lille, France

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

3. CHRU Lille, Service des Maladies de l’appareil digestif

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

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

6.

CHRU Lille, Service de chirurgie endocrinienne

*Corresponding author:

Prof. Philippe Mathurin

2 rue Michel Polonovski

59037, Lille CEDEX

France

Phone : 03 20 44 53 21Email : [email protected]


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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

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

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

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

of lipotoxins that promote hepatic lipogenesis, adipose tissue lipolysis, and impaired β-

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

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

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

3 trials of patients with NASH and early-stage fibrosis1. Although a decrease in the non-

alcoholic fatty liver disease (NAFLD) activity score (NAS) could be used as an endpoint in

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-Pugh-

Turcotte scores, as well as the hepatic venous pressure gradient1. These new approaches

provide a foundation to develop treatments for NAFLD.

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,

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,

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 patient’s 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

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.

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

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

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

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).

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. These

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

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 compliance—especially

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

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

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

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

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 orlistat—an 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 NASH5, 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

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

was stopped due to serious psychiatric side effects—patients developed severe depression and

became suicidal21

. Research is underway to develop a peripherally restricted CB1 antagonist

that reduces liver injury without causing neuropsychiatric events23

.

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

24 and increasing weight loss in patients with type 2 diabetes

25. In addition to the benefits to

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

In severely obese patients (those with a body mass index [BMI] ≥ 40 or 35–40 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

procedure type, bariatric surgery induces long-term weight loss (of 15%–25%), as well as

remission of diabetes29-31

and reduced overall long-term mortality32

—particularly from

diabetes33

, heart disease,34

and cancers35

.

The effects of bariatric surgery on the liver include reductions in steatosis—mainly

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

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

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

reducing liver injury—this 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

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

alter disease progression and prevent development of cirrhosis and its complications.

Cirrhosis is considered to be a contraindication to bariatric surgery—mortality increased 21-

fold in patients with decompensated cirrhosis and 2-fold in patients with compensated

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

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, 46

impairs the ability of insulin to activate glycogen synthesis and

inhibit gluconeogenesis2. These mechanisms have been also been observed in liver tissues

from obese patients with NAFLD; in these patients, hepatic diacylglycerol content,

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 pathway48

. The severity of insulin

resistance was associated with more severe necroinflammation injury in liver tissues from

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 β-oxidation, which worsens oxidative stress. Insulin resistance might be targeted

therapeutically, perhaps with insulin sensitizing agents.

Metformin

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 resistance52

.

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

diabetes57, 58

. Metformin might therefore be used to decrease the risk of liver cancer in

patients with diabetes, but this interesting strategy requires further study58

.

Peroxisome proliferator-activated receptors (PPARs)

Peroxisome proliferator-activated receptors (PPARγ) 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 PPARγ agonists

decrease glucose and glycosylated hemoglobin. Because PPARγ 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.

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

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 non-

diabetic patients with NASH61

compared pioglitazone to placebo, as well as vitamin E to

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

outcome—resolution 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 PPARγ agonists. All large randomized controlled trials of pioglitazone and


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rosiglitzone have shown weight gain of 3–5 kg after patients took these drugs59, 61, 62

.

Furthermore, the FDA and EMA have warned about the cardiovascular risk related to the use

of PPARγ 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

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 PPARα and PPARδ that has been tested in

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

markers of inflammation and fibrogenesis70

. In clinical trials, elafibranor (NCT01271751,

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

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

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

The GLP1 pathway

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 PPARα / γ 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, IL1β, 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

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

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

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

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

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

available.

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

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,

74. In patients with type 2 diabetes and NAFLD, 25 and 50 mg of obeticholic acid per day

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

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,

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 LDL–cholesterol,

but lower levels of HDL–cholesterol. This is a major issue due because these factors increase

the risk of cardiovascular events in this population. Therapeutic strategies that include this

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

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 placebo—not in the groups that received 10 or 20 mg

obeticholic acid77

. The long-term safety and efficacy of the 25 mg/day dose of obeticholic

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

. N‑6 PUFAs, such as linoleic acid and arachidonic acid have been shown

to promote inflammation and synthesis of eicosanoids79. Conversely, n‑3 PUFAs and omega3


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fatty acids, such as α‑linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid

reduce inflammation. In animal models, a diet enriched in n‑3 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 metabolism

80

.The ratio of n-6:n-3 is altered in serum samples from patients with NAFLD

81. A

randomized placebo-controlled trial of 243 patients with NASH or severe NAFLD (NAS

scores of 4 or more) showed that 1.8g–2.4g/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

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

cholanoic acid) lowers the amount of fat in the liver by decreasing synthesis and increasing β

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 β 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

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 β-oxidation87

.

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

general lipodystrophy, a genetic disorder that causes leptin deficiency88, 89

. Data from long-

term studies have shown that although metreleptin significantly reduced steatosis, lobular

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 Niemann–Pick C1–like 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 LDL–cholesterol and improves cardiovascular

outcomes by reducing the risk of myocardial infarction and ischemic stroke90-94

. PCSK9-

knockout 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

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

determine the effects of PCSK9 inhibitors.

Reducing Oxidative Stress

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 β oxidation or endoplasmic reticulum stress, leading to

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

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

other 7 isoforms of vitamin E in patients with NASH. Vitamin E is considered to be the first-

line 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

cirrhosis56

. 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 γ-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

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

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

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

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

mortality.

Non-vitamin E antioxidants

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 NASH56

.

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

inhibiting fibroblasts and decreasing growth factors and the transforming growth factor-β

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 β-galactoside sugars.

Galectin-3 has crosslinking and adhesive properties and is encoded by the lectin, galactoside-

binding, 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

induction of liver fibrosis by toxin administration109

. Phase 1 studies of galectin-3 inhibitors

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).

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

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

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-3–cleaved 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

.

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

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

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.

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

resistance, and liver diseases including NAFLD121, 122

. NASH has been associated with

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

NAFLD and obesity121, 122

. Transfer of intestinal microbiota from lean donors increased

insulin sensitivity in individuals with metabolic syndrome123

. 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

morbidity.


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

Drugs &

Therapy

Action Phase of

development

Histological Impact Pros & cons Ref

Weight loss

Lifestyle Reduces insulin

resistance,cardiovascular

risk; lipid profile reduces

oxidative stress

Recommended in

first-line

treatment

Depends on the level of

weight loss:

▪ >3%–5%: reduces

steatosis

▪ > 7%–9%: reduces

necroinflammation

▪ ≥10%: reduces fibrosis

Only 10% of patients reach the

10% weight loss threshold

required for an effect, and risk

weight gain relapse

6, 7, 56

Bariatric surgery Induces sustained weight

loss at least up to 10 years;

increases insulin resistance

and cardiovascular risk

Cohort studies Reverses NASH in 85%

of patients. Reduces

steatosis,

necroinflammation, and

fibrosis

Recommended for patients

with morbid obesity. Post-

operative morbidity limits

applicability to patients with

BMIs ≤ 35kg/m².

37-39

Insulin sensitizers

Pioglitazone PPARγ agonist, lipid

homeostasis, inflammation,

and cell differentiation

Phase 2

completed

Improves:

▪ steatosis

▪ necroinflammation

▪ fibrosis

Pros: effective for patients

with NASH

Cons: Weight gain

Risk of congestive heart

failure (no effect on mortality)

and bladder cancer

59, 61

Liraglutide GLP1 agonist, promotes

satiation, weight loss,

reduces insulin resistance

Phase 3 Reduces

▪ steatosis


No worsening of fibrosis

▪ resolution of NASH

Pros: marketed for patients

with diabetes, safety seems

acceptable

Cons: gastrointestinal side

effects, requires subcutaneous

administration

26

Agents with multiple targets

GFT505/

Elfibranor

PPARα and PPARδ

agonist;

reduces insulin resistance,

inflammation, and fibrosis;

improves lipid metabolism

Phase 3 Reduces

▪ steatosis

▪ necroinfammation

▪ fibrosis

Reverses NASH *

120 mg per day reduces

histologic features of NASH

65, 126

Obeticholic acid FXR agonist, increases

energy homeostasis and

lipid metabolism; reduces

insulin resistance,

inflammation, and fibrotic

pathways

Phase 3 Reduces

▪ steatosis


▪ fibrosis

▪ NAS without worsening

of fibrosis

Pros: reduces histologic

features in NASH.

Cons: side effects, with 20%

of patients developing pruritus

72,73

Agents that alter lipid metabolism

Aramchol Inhibition of stearoyl

coenzyme A desaturase 1

Phase 2 Decrease fat amount, no

effects on NASH

No effects on NASH, only

preliminary results available

83


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(SD1)

Anti-oxidants

Vitamin E Tocopherol, anti-oxydative

stress

Phase 2

completed

Significant effects on

▪ steatosis


▪ NAS

Pros: recommended as first

pharmacologic therapy

Cons: no data from patients

with diabetes or cirrhosis;

concerns for long-term use

1

Agents that reduce inflammation or cell death

Pentoxifylline Reduces inflammation

(potentially by inhibiting

TNF), blood viscosity, and

platelet aggregation

Phase 2

completed

Reduces

▪ steatosis


▪ NAS

Pros: pilot studies indicate

efficacy

Cons: lack of large

randomized controlled trials or

robust data sets

11

Cenicriviroc Inhibit CCR2 and

CCR5, reduces

insulin resistance and

recruitment of

inflammatory cells

Phase 2 No data Initially developed for

treatment of HIV infection.

Potentially interesting effects

in patients with NASH but

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

controlled trials.


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Table 2. Agents in Development

Drug Target Potential Mechanism of

Action

ClinicalTrial.gov

identifier

GR-MD-02 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

oxidative stress

NCT02466516

JKB-121 Toll-like receptor 4 Anti-inflammatory agent NCT02442687

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


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Figure 1. Pathophysiology of NASH and Therapeutic Targets

Legend:

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

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.

Fat absorption may be targeted to reduce steatosis, as for example NCP1L1 (Niemann–Pick

C1–like 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

activity. Hepatic insulin resistance increases lipogenesis and reduces fatty acids β-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 β-oxidation and reduced lipogenesis. In the therapeutic landscape for NASH, many

agents target insulin resistance (liraglutide, PPARα 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,

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

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


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2016 Perspectives on Treatment for Nonalcoholic Steatohepatitis. GASTROENT

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