Drug Discovery Today d Volume 29, Number 3 d March 2024 REVIEWS

KEYNOTE (GREEN)
NAFLD and NASH: etiology, targets and
emerging therapies
Shulin Wei 1,2, Li Wang 3,
Paul C. Evans 4, Suowen Xu 2,⇑
Suowen Xu is a Professor of Pharmacology at
the University of Science and Technology of
China in Hefei and a visiting professor at the
Max Planck Institute for Heart and Lung
1 Research in Bad Nauheim, Germany. He was
School of Life Sciences, Jilin University, Changchun, China
2
Department of Endocrinology, Institute of Endocrine and Metabolic Disease, selected for China’s High-level Special Talent
Program and Young Top Talent Program, and
The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, has been listed among the 2022 world’s top 2%
Clinical Research Hospital of Chinese Academy of Sciences (Hefei), of scientists (Elsevier-Standford University list).
University of Science and Technology of China, Hefei, China He received his PhD in pharmacology from Sun
3
Department of Biomedical Sciences, City University of Hong Kong, China Yat-sen University in Guangzhou, China, in
4 2011, and he conducted his postdoctoral
Centre for Biochemical Pharmacology, William Harvey Research Institute,
research at the National Institutes of Health
Barts and The London Faculty of Medicine and Dentistry,
(NIH) in Bethesda, Maryland, and the University of Rochester in New York
Queen Mary University of London, EC1M 6BQ, UK State between 2011 and 2016. He was promoted to research assistant
professor at the University of Rochester and held the position of adjunct
associate professor at the University of Queensland in Brisbane, Australia. He
has long been engaged in researching the pathogenesis and pharma-
Non-alcoholic fatty liver disease (NAFLD) and non- cotherapies of atherosclerosis, diabesity and fatty liver diseases. He has been
funded by the American Heart Association (AHA) and National Natural Sci-
alcoholic steatohepatitis (NASH) pose a significant threat ence Foundation of China, and is the recipient of a Career Development
Award from the AHA and a Humboldt Fellowship for Experienced
to human health and cause a tremendous socioeconomic Researchers from the Alexander von Humboldt Foundation in Germany.
burden. Currently, the molecular mechanisms of NAFLD
and NASH remain incompletely understood, and no
effective pharmacotherapies have been approved. In the
past five years, significant advances have been achieved in
our understanding of the pathomechanisms and potential
pharmacotherapies of NAFLD and NASH. Research
advances include the investigation of the effects of the
fibroblast growth factor 21 (FGF21) analog pegozafermin
and the thyroid hormone receptor-b (THRb) agonist res-
metriom on hepatic fat content, NASH resolution and/or
fibrosis regression. Future directions of NAFLD and NASH
research (including combination therapy, organoids and
humanized mouse models) are also discussed in this state-
of-the-art review.

⇑ Corresponding author. Xu, S. ([email protected])

1359-6446/Ó 2024 Elsevier Ltd. All rights reserved.

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 KEYNOTE (GREEN) Drug Discovery Today d Volume 29, Number 3 d March 2024

Introduction state results in inflammation, insulin resistance and increased

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent levels of free fatty acids (FFAs). Furthermore, excessive FFA
chronic liver disease worldwide, and its disease spectrum encom- uptake contributes to the development of lipotoxicity. Mean-
passes non-alcoholic fatty liver (NAFL), non-alcoholic steatohep- while, increased production of reactive oxygen species (ROS)
atitis (NASH), liver cirrhosis and hepatocellular carcinoma induces apoptosis (and other types of cell death) and amplifies
(HCC). NAFL is characterized by macrovesicular steatosis inflammation, thus inflicting the second hit on the liver(p10).
in  5% of hepatocytes with no hepatocellular damage(p1). As a As research has progressed, additional factors such as genetic sus-
result of the minimal inflammation present in patients with ceptibility, endoplasmic reticulum (ER) stress, intestinal flora
NAFL, they can recover through lifestyle interventions such as alteration, oxidative stress and hepatic stellate cell (HSC) activa-
increased physical activity or dietary alterations. NASH, an tion have been incorporated into the mechanism of NAFLD/
KEYNOTE (GREEN)

advanced stage of NAFLD, involves hepatocyte ballooning, insu- NASH disease progression (Figure 1). The following sections elab-
lin resistance and lobular inflammation(p2). The gold standard for orate on the contributions of these factors to the disease.
diagnosing NASH and differentiating it from NAFL remains liver
biopsy. Steatosis activity fibrosis (SAF) and the NAFLD activity
score (NAS) are the current evaluation criteria for NASH staging, Dietary habits
with NAS being the more widely used scoring system. NASH can Dietary habits could influence the advance of NAFLD and NASH
be diagnosed when the histological characteristics of steatosis (0– in different ways. For example, resistant starch decreases levels of
3), hepatocyte ballooning (0–2), lobular inflammation (0–2) and triglycerides (TGs), which can improve NAFLD, and calorie
fibrosis (0–4) culminate in a NAS score equal to or above 5(p2). restriction induces weight loss(p11),(p12). The western diet, which
NASH can deteriorate into cirrhosis or HCC without appropriate is low in choline and high in sugar and fat, induced the produc-
and timely interventions(p3). tion of 2-oleoylglycerol in western-diet-fed mice, which con-
NAFLD is increasing in prevalence. The global incidence of tributed to inflammation and fibrosis in the liver(p13).
NAFLD is currently estimated to be 38% in the general popula- Moreover, high-starch carbohydrates can worsen NAFLD by
tion, with much higher rates among overweight and obese pop- enhancing fatty acid influx and inducing increased intrahepatic
ulations(p4),(p5). Furthermore, the liver is often considered as a expression of NADPH oxidase 2 (NOX2)(p14).
‘silent’ organ, displaying no discernible clinical symptoms or dis-
comfort until a diagnosis of liver cirrhosis(p6). This serves as
another key factor leading to the rising incidence of NAFLD. Metabolic dysfunction of hepatocytes
Patients with NAFLD and NASH might experience complications Excessive accumulation of TGs in hepatocytes is the major
that further exacerbate the risks to those affected. Therefore, pathophysiological characteristic of NAFLD, which suggests that
urgent extensive research is warranted to unravel the pathologi- disordered metabolism of TGs or other lipids is linked to the
cal mechanisms of NAFLD and to identify effective pharma- occurrence and development of NAFLD and NASH. For instance,
cotherapies to mitigate these harmful threats. sterol-regulatory element binding protein (SREBP), which is
One question that should be addressed is the name NAFLD. responsible for cholesterol and TG synthesis, is abnormally acti-
The word ‘fatty’ is stigmatized to some extent. Therefore, a con- vated in patients with NASH. Inhibiting SREBP activation by
sensus group chose to replace NAFLD with metabolic binding insulin-induced genes offers potential therapeutic
dysfunction-associated steatotic liver disease (MASLD) in effects in the treatment of NAFLD(p15). In addition, a recent study
2023(p7). Interestingly, by using MASLD, metabolic associated showed that EH domain binding protein 1 (EHBP1), a
fatty liver disease (MAFLD), and NAFLD to define subjects, it cholesterol-associated loci, is enriched in human NASH, resulting
was found that the differences between MASLD and NAFLD were in weakened low-density lipoprotein (LDL) receptor degradation
so small that the previous NAFLD data could be applied to and increased levels of transcriptional co-activator with PDZ-
MASLD(p8). However, considering that most of the current arti- binding motif (TAZ), which exacerbates NASH(p16).
cles and clinical trials on this disease focus on NAFLD, and that
the clinical trials conducted on patients diagnosed with MASLD
are limited, we use the term NAFLD throughout this review. This Gut microorganisms
article is aimed to review up-to-date translational advances in Numerous studies have shown that the gut microbiome and its
potential pharmacotherapies for NAFLD and NASH, and it pro- components have a substantial role in human health: for exam-
vides a mechanistic perspective on these pharmacological ple, gut microorganisms could alleviate NASH through nicotine
actions. degradation, and levels of histidine (an energy source for gut
microorganisms) are inversely related to NAFLD develop-
ment(p17),(p18). Pathogen-associated molecular patterns (PAMPs)
NASH pathogenesis: A brief review such as lipopolysaccharide (LPS) are conserved molecular struc-
Early studies proposed a ‘double-hit’ theory describing the patho- tures that are shared by pathogenic microorganisms, and LPS is
genesis of NAFLD. The initial ‘hit’ can be explained by impaired the main component of the outer membrane of Gram-negative
lipid metabolism, where liver parenchymal cells accumulate bacteria. It induces inflammatory cytokine activation and intesti-
excessive triacylglycerol in lipid droplets, subsequently causing nal barrier impairment, following which intestinal permeability
hepatic steatosis(p9). The subsequent alteration of macrophages increases and LPS enters into the liver, further aggravating
from an anti-inflammatory to a proinflammatory phenotypic NAFLD(p19).

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FIGURE 1
Complex mechanisms driving the pathological development of NAFLD. Dietary habits such as a high fat and high fructose diet could induce DNL and fatty
acid influx, thus leading to lipid accumulation and insulin resistance. The gut microbiota and their metabolites can promote gut dysbiosis, DNL, inflammation
and steatosis. Apart from that, NAFLD is inherited, and several susceptibility loci also contribute to the pathological progression of NAFLD.

Genetic susceptibility and epigenetic mechanisms increased level of C3 but not C4 was linked to NAFLD by mende-
Genetic factors are also pivotal in NAFLD and NASH, with 35– lian randomization analysis(p25). In addition, tripartite motif
61% of cases potentially being inherited. Five potential suscepti- (TRIM) family proteins are essential in natural immune responses
bility loci have been identified: glucokinase regulator (GCKR), against viral infections, and TRIM56 induces fatty acid synthase
tribbles pseudokinase 1 (TRIB1), transmembrane 6 superfamily (FASN) degradation to delay the progression of NAFLD(p26). NLR
member 2 (TM6SF2), apolipoprotein E (APOE) and patatin-like family pyrin domain containing 3 (NLRP3) can also promote
phospholipase domain-containing protein 3 (PNPLA3)(p20). liver inflammation through myeloid cells, and NLPR3-knockout
Recently, it was found that homozygous PNPLA3-I148M causes mice showed decreased fibrosis(p27). Deficiencies in cyclic GMP-
hepatic mitochondrial dysfunction and decreased de novo lipoge- AMP synthase (cGAS), a DNA sensor that participates in inflam-
nesis (DNL)(p21). In addition, rare ATG7 mutations could acceler- mation, were shown to aggravate steatosis and inflammation in
ate NAFLD progression through increased ballooning and mice in which NASH was induced through a diet high in fat,
inflammation(p22). Furthermore, one protective variant in hydrox- sugar and cholesterol(p28).
ysteroid 17-beta-dehydrogenase 13 (HSD17B13) has been identified
recently. HSD17B13, a hepatocyte-specific protein, has been Oxidative stress and ER stress
implicated in the initiation and progression of NAFLD and NASH NAFLD could be accelerated by oxidative stress triggered through
in both mice and humans. A splice variant in HSD17B13 was the increased generation of ROS (a natural byproduct of aerobic
associated with mitigated histological characteristics of NAFLD metabolism). ROS can exacerbate hepatic fibrosis by activating
and reduced SAF score in patients(p23). Epigenetic modifications, HSCs to produce more extracellular matrix(p29). In addition to
such as microRNAs (miRNAs), promote the pathological progres- the direct role of ROS in fibrogenesis, ROS serves as an important
sion of NAFLD alongside genetic alterations. For example, sup- regulator of NF-jB activity, which aggravates NAFLD via immune
pression of miR-145a-5p promotes NAFLD progression through and inflammatory pathways(p30). In addition, the production of
enhanced inflammation and lipid accumulation in mice(p24). oxidized phospholipid (OxPL) as a result of oxidative stress leads
to mitochondrial dysfunction in mice hepatocytes through ROS
Immune system and inflammatory signaling pathways accumulation, whereas neutralizing OxPL relieves oxidative
Patients with NAFLD and NASH exhibit chronic liver inflamma- stress, inflammation and fibrosis(p31). Excessive ROS generation
tion and disordered inflammatory pathways. For instance, com- also leads to ER stress. Recently, it was found that higher levels
plement 3 (C3) and C4) are proteins that participate in of forkhead box A3 (FOXA3) led to NAFLD progression through
immune and inflammatory responses. It was found that an enhanced ER stress in mice(p32).

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 KEYNOTE (GREEN) Drug Discovery Today d Volume 29, Number 3 d March 2024

Non-pharmacological interventions: Bariatric surgery restricting caloric intake might produce more effects in terms
of NAFLD management when coupled with time-restricted
and lifestyle modification
eating(p35).
NAFLD is primarily associated with abnormal accumulation of
Bariatric surgery offers another non-pharmacological treat-
fat in hepatocytes on account of various factors, excluding alco-
ment for individuals with NAFLD(p36). A 2022 systematic review
hol and other explicit injury factors. The first-line approach for
encompassing 37 studies indicated that bariatric surgery resulted
treating NAFLD includes lifestyle adjustments, such as embark-
in improvement in steatosis in 56% of patients, improved bal-
ing on a low-calorie diet, increasing levels of physical exercise
looning in 49% of patients, and reduced inflammation in 45%
and reducing alcohol consumption (Figure 2). The goal of life-
of patients, and the effect was more pronounced among patients
style modification is weight loss, which has a large role in the
from Asian countries(p37). Notably, it was found that bariatric sur-
KEYNOTE (GREEN)

improvement of NAFLD. Engaging in aerobic and resistance

gery produced significant reduction in cardiovascular disease
training for 150 to 200 minutes per week is recommended(p33).
(CVD) risk in severe obese individuals and patients with
An early follow-up study showed that 12 months of regular vig-
NAFLD(p38).
orous or moderate exercise led to reduced abdominal obesity,
However, the effect of bariatric surgery and lifestyle modifica-
lower blood pressure and lower levels of intrahepatic TG con-
tion are restricted by poor execution, low compliance and insuf-
tent(p34). In addition to physical activity, dietary modifications
ficient clinical data. In addition, a ‘rebound’ effect can be set off
can be a valuable option. A randomized trial suggested that

Drug Discovery Today

FIGURE 2
Effects of bariatric surgery and lifestyle modification and on NAFLD. Non-pharmacological strategies such as bariatric surgery and lifestyle modification could
both improve NAFLD by affecting weight loss, insulin resistance, hepatic steatosis, ballooning, fibrosis and the levels of biomarkers associated with the
disease. Lifestyle modifications can also offer benefits in terms of reducing anxiety, CVD risk, portal hypertension, blood pressure and fatty acid oxidation.
Abbreviations: FA, fatty acid.

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FIGURE 3
Potential molecular targets and targeted pharmacotherapies for NAFLD and NASH. According to the ‘multiple-hit theory’ for the pathogenesis of NAFLD,
there are various molecular targets for disease intervention. Most pharmacotherapies focus on abnormal lipid accumulation, insulin resistance, fibrogenesis
and inflammation. Specific drug categories are listed underneath each pathological event.

after patients gain weight again. In practice, the efficacy of Peroxisome proliferator-activated receptor agonists
lifestyle, activity and dietary changes and even surgical interven- Three isoforms of peroxisome proliferator-activated receptors
tions might not be high and prolonged. Therefore, there should (PPARs) have been identified: a, b/d and c. PPARa is highly
be a focus on utilizing pharmacotherapies or the combining of expressed in the liver, and its activation facilitates fatty acid oxi-
non-pharmacological strategies with novel pharmacotherapies. dation and degradation, inflammatory response inhibition and
insulin sensitivity improvement. The activation of PPARb/d has
Pharmacotherapies in clinical development for also shown promise by improving insulin resistance. PPARc ago-
treating NASH nists stimulate lipolysis and enhance insulin sensitivity(p39).
Recently, extensive research has shed light on multiple NASH Given the physiological role of PPARs, it could be inferred that
pathological mechanisms, but no FDA or EMA-approved drug modulation of PPAR might offer positive effects in treating
exists for treating NASH as a result of the undesirable side effects NASH. In this regard, saroglitazar acts as a dual agonist for PPARa
or inadequate clinical end-point efficacy of the drugs trialed so and PPARc. The primary endpoint of a phase II trial was to reduce
far. However, there is a glimmer of hope in the form of the sub- the level of alanine transaminase (ALT). The primary endpoint
stantial influx of newly developed drugs in the pipeline, many of was reached in 1 mg dose (-27.3%), 2 mg dose (–33.1%) and
which promise to become pioneering NAFLD/NASH treatments. 4 mg dose (-44.3%) groups versus the placebo group (4.1%),
Present clinical trials commonly measure two main outcomes: and the 4 mg group exhibited a notable decrease in liver fat con-
(i) NASH resolution without worsening fibrosis and (ii) fibrosis tent, TGs and cholesterol levels (NCT03061721)(p40). Currently, a
regression without worsening NASH. In terms of available phar- phase IV clinical trial aimed at patients with NAFLD and obesity,
macotherapies, monotherapies can be broadly classified into type 2 diabetes mellitus (T2DM), dyslipidemia or metabolic syn-
groups such as metabolic, anti-inflammatory and antifibrotic drome is ongoing (NCT05872269).
(Figures 3 and 4). Furthermore, various combination therapies Elafibranor activates both PPARa and PPARb. However, its
have been explored to optimize the therapeutic efficacy and/or development was terminated owing to its inability to achieve
reduce drug side effects. This paper presents a state-of-the-art NASH resolution with no worsened fibrosis in a phase III trial
summary of the targets and current clinical research progress (NCT02704403). However, lanifibranor, which activates PPARa,
on newly developed or applied medications (Table 1). PPARd and PPARc, has shown promise in a phase IIb trial, with

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FIGURE 4
Potential pharmacotherapies for treating NAFLD and NASH. Specific candidate drugs are listed under each drug category. In the GLP1R/GCGR/GIPR agonists
category, semaglutide is a GLP1R agonist, cotadutide is a dual agonist for GLP1R and GCGR, efinopegdutide is a dual agonist for GLP1R and GCGR, tirzepatide
is a dual agonist for GLP1R and GIPR, and retatrutide is a triple agonist for GLP1R, GCGR and GIPR.

a higher ratio of  2-point reduction in the SAF-A score without Resmetriom serves as a selective THRb agonist, and the phase
fibrosis worsening compared with the placebo group II clinical trial suggested statistically significantly decreases in
(NCT03008070)(p41). Phase II (NCT03459079) and III liver fat, LDL cholesterol (LDL-C) and ApoB in the treated group,
(NCT04849728) trials evaluating the efficacy and safety of lanifi- along with improved fibrosis (NCT02912260)(p44). Currently,
branor are recruiting patients, and lanifibranor received a break- four phase III clinical trials of resmetriom are ongoing. The
through therapy designation from the FDA. MAESTRO-NASH trial focused on patients with biopsy-
Pemafibrate acts as a selective PPARa modulator (SPPARMa) confirmed NASH, where the 80 mg and 100 mg groups achieved
and shows improvements in both mice and human studies. In both primary endpoints and the key secondary endpoint. The
a randomized phase II trial, although pemafibrate did not reach diagnosis in MAESTRO-NAFLD-1 was based on non-invasive
significance in the primary endpoint, it reduced liver stiffness measures instead of liver biopsy. The primary and secondary end-
and ALT level with good tolerability (NCT03350165)(p42). points focused on safety and tolerability, as well as the reduction
of LDL-C, ApoB, hepatic fat fraction and TGs. Both endpoints
were achieved(p45). Phase I, II and III tests have provided conclu-
Thyroid hormone receptor-b agonists
sive data supporting a priority review of the new drug application
Thyroid hormone receptor (THR) consists of two isoforms: THRa,
for resmetriom for the treatment of NASH. TG68, another THRb
which is highly expressed in cardiac as well as skeletal muscle tis-
agonist, has been shown to decrease liver volume, improve
sues, where activating it might cause side effects in the bone and
steatosis and mitigate hepatic damage in high-fat-diet-treated
heart; and THRb, which is primarily distributed in the liver and
mice(p46). Further clinical data are necessary for validating these
crucially takes part in lipid metabolism(p43). Therefore, activating
findings.
THRb selectively might offer a promising therapeutic approach
for NASH while minimizing the side effects of global THR activa-
tion. VK2809, one of the THRb agonists, was investigated for its Fibroblast growth factor 21 analogs
anti-NAFLD efficacy and safety in a phase II trial, but no results The fibroblast growth factor (FGF) family is comprised of diverse
have been posted (NCT02927184). growth factors performing various functions. Notably, the FGF19

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TABLE 1
Drugs in clinical trials for NAFLD and NASH treatment
Class Drug Mechanism Cohort Phase NCT number Reference
(p40)
PPAR agonists Saroglitazar Activates PPAR a/c; NAFLD/NASH II NCT03061721
Improves insulin sensitivity. NAFLD with IV NCT05872269 NA
Comorbidities
Elafibranor Activates PPAR a/d; NASH and fibrosis III NCT02704403 NA
Improves insulin sensitivity;
Improves inflammation;
Enhances fatty acid oxidation;

KEYNOTE (GREEN)
(p42)
Pemafibrate Activates PPAR a; NAFLD II NCT03350165
Enhances FFA metabolism;
Improves atherogenic dyslipidaemia.
(p41)
Lanifibranor Activates PPAR a/d/c; NASH II NCT030080708
Improves glucose and FFA metabolism. T2MD and NAFLD II NCT03459079 NA
NASH and F2/3 III NCT04849728 NA
THR-b agonists VK2809 Reduces plasma and liver lipids; Primary II NCT02927184 NA
Improves liver fibrosis. hypercholesterolemia
and NAFLD
(p44)
Resmetriom Activates THR-b in the liver selectively; NASH II NCT02912260
Promotes liver fat decomposition; NASH and fibrosis III NCT03900429 NA
(p45)
NAFLD III NCT04197479
FGF19 analogues NGM282 Reduces liver fat content and ALT NASH II NCT02443116 NA
content; Compensated cirrhosis II NCT04210245 NA
Reduces expression of profibrotic
genes.
(p86)
FGF1R agonists BFKB8488 Activate FGF1R and Klothob. NAFLD and T2MD I NCT03060538
(p48)
FGF21 analogues Pegozafermin Stimulates FGF21 for long term; NASH or NAFLD I/II NCT04048135
(p49)
Improves insulin resistance; NASH II NCT04929483
Reduces serum lipids and body weight.
(p50)
Pegbelfermin Activates FGFs; NASH II NCT02413372
Reduces oxidation stress and ER stress; NASH III NCT03400163 NA
Improves chronic inflammation.
(p51)
Efruxifermin Stimulates the agonisms of FGFR1c, NASH II NCT04767529
FGFR2c and FGFR3c.

(p53)
SCD1 inhibitors Aramchol Reduces DNL; NASH II NCT02279524
Enhances fatty acid oxidation. NASH III NCT04104321 NA
(p55)
ASK1 inhibitors Selonsertib Inhibits the formation of inflammatory NASH and III NCT03053050
cytokines; bridging fibrosis
(p55)
Reduces the gene expression related to NASH III NCT03053063
fibrosis;
Inhibits excessive apoptosis.
(p56)
FASN inhibitors FT-4101 Inhibits DNL; NASH I/II NCT04004325
Improves hepatic steatosis;
(p57)
TVB-2640 Inhibits DNL; NASH II NCT03938246
Improves fibrosis and inflammation.
DGAT1/2 inhibitors Pradigastat Inhibits DGAT1; NAFLD II NCT01811472 NA
Reduces liver fat content and TGs.
(p58)
PF-06865571 Inhibits DGAT2; NAFLD I NCT03513588
Inhibits DNL.
(p62)
MPC inhibitors MSDC-0602K Binds to MPC and regulates the influx of NASH II NCT02784444
pyruvic acid;
Increases insulin sensitivity.
(p67)
FXR agonists Tropifexor Reduces lipid synthesis; NASH II NCT02855164
Improves fibrosis and steatosis. NASH II NCT03421431 (p68)
(p65)
OCA Increases insulin sensitivity; NASH III NCT02548351
Improves hepatocyte cytotoxicity;
Improves inflammation in the liver.
EDP-305 Inhibits lipogenesis; NASH IIb NCT04378010 NA
Improves inflammation and steatosis;
Reduces liver injury and fibrosis.
Cilofexor Reduces liver fibrosis; Primary sclerosing III NCT03890120 (p66)

Reduces intrahepatic sinusoidal cholangitis

resistance; Inhibits fibrogenesis.
HEC96719 Activates FXR. Healthy subjects I NCT04422496 NA
(continued on next page)

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TABLE 1 (CONTINUED)
Class Drug Mechanism Cohort Phase NCT number Reference
(p71)
GLP1R/GCGR/GIPR Cotadutide Reduces lipid accumulation; T2DM II NCT03555994
agonists Improves inflammation and fibrosis.
(p70)
Semaglutide Increase insulin secretion and NASH II NCT02970942
sensitivity; Non-cirrhotic NASH III NCT04822181 NA
Inhibits the release of glucagon;
Inhibits hepatic gluconeogenesis.
(p72)
Efinopegdutide Activates both GLP1R/GLP. NAFLD II NCT04944992
Hepatic impairment I NCT06052566 NA
(p73)
Tirzepatide Activates both GLP1R/GIP; T2DM II NCT03131687
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Increases insulin secretion. NASH II NCT04166773 NA

SGLT2 inhibitors Dapagliflozin Reduces steatosis and fibrosis. NASH III NCT03723252 NA
ACLY inhibitors Bempedoic Upregulates LDL receptor; NAFLD and T2DM Not NCT06035874 NA
acid Reduces the synthesis of cholesterol. Applicable
(p88)
LXR inhibitors Oltipraz Inhibits LXR-a； NAFLD except liver II NCT01373554
Inhibits the lipid synthesis. cirrhosis
(p89)
CCR2/5 inhibitors Cenicriviroc Inhibits both CCR2 and CCR5; NASH III NCT03028740
Improves inflammation and fibrosis.
(p83)
ACC GS-0976 Inhibits both ACC1 and ACC2; NASH II NCT02856555
inhibitors Suppresses DNL.
Clesacostat Reduces liver fat; NAFLD II NCT03248882 NA
Reduces ALT content.
A3AR agonist Namodenoson Reduces inflammation; NASH II NCT04697810 NA
Improves fibrosis.
(p90)
AOC3 inhibitor BI 1467335 Reduces ALT content. NASH II NCT03166735
(p91)
Pan-PDE inhibitor ZSP1601 Reduces liver fat content; NASH I/II NCT04140123
Improves liver fibrosis.
AMPK activators PXL770 Reduces liver fat; NAFLD II NCT03763877 NA
Reduces HbA1c.
(p93)
HSD17B13 inhibitors ARO-HSD Reduces HSD17B13 and ALT; NASH and healthy I NCT04202354
Inhibits HSD17B13. individuals
GSK4532990 Inhibits HSD17B13. NASH and F3 II NCT05583344 NA
INI-822 Inhibits HSD17B13. NASH and healthy I NCT05945537 NA
individuals

subfamily, which includes FGF19, FGF21 and FGF23, has been has promising potential for the treatment of F2 or F3 patients
strongly implicated in metabolic diseases. FGF21 is pivotal in and warrants further evaluation in a phase III trial.
metabolism because it enhances lipid uptake in adipose tissue
and prevents hepatic lipid accumulation(p47). Pegozafermin, serv- Stearoyl-CoA desaturase 1 inhibitors
ing as a long-acting glycopegylated FGF21 analog, has been Stearoyl-CoA desaturase 1 (SCD1) catalyzes the rate-limiting step
applied for the treatment of severe hypertriglyceridemia and in producing monounsaturated fatty acids (MUFAs). Several clin-
NASH. A phase Ib/IIa dose-ascending study on subjects with ical trials have indicated that knocking down the SCD1 gene or
NASH and F1–F3 fibrosis, or NAFLD and a high risk of NASH, inhibiting SCD1 activity could reduce hepatic lipid accumulation
indicated that pegozafermin significantly reduced liver fat with and lipid toxicity(p52). Aramchol is one example of an SCD1 inhi-
good tolerance and mild adverse events (NCT04048135)(p48). In bitor. Data from a phase IIb trial show that the group treated
a phase IIb trial, there was a dose-dependent percentage in reach- with aramchol at a dose of 600 mg (16.7%) reached NASH reso-
ing primary endpoint except for NASH resolution lution without fibrosis worsening when compared with the pla-
(NCT04929483)(p49), which supports the advancement of cebo group (5%). Furthermore, both the 400 mg and 600 mg
pegozafermin into phase III clinical trials. treatment groups showed reduced levels of liver fat, ALT and
Another analog of FGF21, pegbelfermin, reduced the mean aspartate transaminase (AST), with a low incidence of severe
hepatic fat fraction in patients with NAFLD and was well- adverse events (NCT02279524)(p53). Aramchol is currently under-
tolerated (NCT02413372)(p50). Additionally, efruxifermin is a going a phase III trial to investigate its safety and tolerability
bivalent analog of Fc-FGF21. In a phase IIb trial, a one or more (NCT04104321).
stage improvement in liver fibrosis was attained in the 50 mg
dose (41%) and 28 mg dose (39%) groups when compared with Apoptosis signal-regulating kinase 1 inhibitors
the placebo group (20%), and the achievement of primary end- Apoptosis signal-regulating kinase 1 (ASK1) is an important
point was up to 70% in the 50 mg group at week 24. The most kinase that is activated by oxidative stress, inflammatory signals,
common adverse effects were diarrhea and nausea ER stress and related stimuli. ASK1 aggravates lipotoxicity, steato-
(NCT04767529)(p51). These results indicate that efruxifermin sis and insulin resistance, whereas ASK1 inhibition leads to

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Drug Discovery Today d Volume 29, Number 3 d March 2024 KEYNOTE (GREEN)

reduced liver cell death and fibrogenesis in mice(p54). Selonsertib, insulin resistance. Subjects treated with metformin in a random-
an inhibitor of ASK1, is currently being evaluated in two phase III ized trial exhibited lower HbA1c levels and intrahepatic fat con-
trials against NASH with bridging fibrosis or compensated cirrho- tent, and showed improved liver function and weight loss(p60).
sis (NCT03053050; NCT03053063). However, no statistical dif- However, the deficiency in clinical data and the necessity to
ferences were found in fibrosis regression between the drug- improve NAFLD histology make the utilization of metformin in
treated and placebo groups according to the interim data(p55). treating NAFLD and NASH uncertain. Another AMPK activator,
These outcomes have resulted in the re-evaluation and termina- PXL770, did not meet the primary endpoint but was well toler-
tion of this strategy, and suggest that this compound alone is ated in a phase II trial (NCT03763877).
unlikely to achieve a therapeutic effect in treating NASH.
Mitochondrial pyruvate carrier inhibitors

KEYNOTE (GREEN)
Fatty acid synthase inhibitors Mitochondrial pyruvate carrier (MPC) is situated in the inner
An abnormal and ectopic accumulation of lipids contributes to membrane of mitochondria and facilitates the transportation of
the onset and progression of NAFLD and NASH. Fatty acid syn- pyruvic acid. Pyruvic acid participates in various metabolic pro-
thase (FASN) is a crucial enzyme that is involved in the process cesses and provides the body with energy(p61). MSDC-0602 K acts
of DNL and has robust regulatory abilities. Therefore, inhibiting as an MPC inhibitor and insulin sensitizer. In a phase II trial, the
its activity could aid in decreasing DNL and lipid accumulation. results suggested that the primary endpoint was not statistically
FT-4101 inhibited DNL and demonstrated improved hepatic achieved, but the outcome was dose-dependent after adminis-
steatosis in a phase I/II trial (NCT04004325)(p56), but the devel- trating MSDC-0602 K (NCT02784444)(p62). MSDC-0602 K was
opment of FT-4101 has been terminated. A phase II trial indi- well tolerated, which justifies advancing it to a phase III trial in
cated that TVB-2640, another FASN inhibitor, significantly patients with T2DM or T2DM coexisting with NAFLD or NASH
lowered liver fat content, and 61% of the high dose group (NCT03970031).
achieved a minimum reduction of 30% in liver fat content
(NCT03938246). In addition, TVB-260 demonstrated a high Farnesoid X receptor agonists
degree of tolerance with minimal incidence of only mild side Farnesoid X receptor (FXR) is prevalent in both the intestines and
effects(p57). All of these favorable outcomes provide substantial liver, and potentially has a role in inflammation or fibrosis. Acti-
motivation for initiating another phase IIb trial (NCT04906421). vating FXR lowers the levels of hepatic fatty acids in mice and
humans through repressing the expression of lipid metabolism-
Diacylglycerol acyltransferase 1 and 2 inhibitors related enzymes and inhibiting lipid absorption(p63). In addition,
TG is the main lipid that is accumulated in the liver. TG synthe- FXR activation has the added benefit of reducing inflammation
sis is a complex process that is required for multiple enzymes, and fibrosis in obese diabetic mice(p64).
with diacylglycerol acyltransferase (DGAT) facilitating the final Several FXR agonists have undergone clinical trials, including
metabolic step. In mammals, two isoforms of DGAT, namely tropifexor, EDP305, cilofexor and obeticholic acid (OCA). OCA
DGAT1 and DGAT2, have distinct roles in lipid metabolism. efficiently activates FXR as a bile acid analog and represents the
DGAT1 re-esterifies diacylglycerides derived from lipase- first FXR agonist to enter clinical phase III trials. In the phase
mediated hydrolysis, whereas DGAT2 participates in endogenous III REGENERATE study, 22.4% of patients in the 25 mg dose
fatty acid synthesis(p58). A phase II trial evaluated the safety, group achieved fibrosis regression by one or more stages without
effectiveness and tolerability of pradigastat, a DGAT1 inhibitor. worsened NASH, compared with only 9.6% of patients in the pla-
The results showed that both the high-dose group (35.29%) cebo group(p65). However, because of safety concerns and because
and low-dose group (30%) achieved a liver fat content reduction it achieved only one of the primary endpoints, the FDA rejected
of at least 30%, whereas the placebo group experienced a fre- the new drug application for OCA. Cilofexor is a non-steroidal
quency of only 10.53%. In addition, level of fasting TGs was sig- FXR agonist, and in a phase III test targeting patients with pri-
nificantly decreased in pradigastat-treated groups mary sclerosing cholangitis (a rare, chronic cholestatic liver dis-
(NCT01811472). Administration of a DGAT2 inhibitor, PF- ease featuring intrahepatic or extrahepatic stricturing) but no
06865571, resulted in a statistically significant reduction in cirrhosis, less than 10% of participants achieved the primary
whole liver fat content detected by micromagnetic resonance endpoint, leading to trial termination (NCT03890120)(p66). A
imaging. However, the drug-treated group showed a higher inci- phase II trial for tropifexor found that the drug-treated group
dence of adverse effects (NCT03513588)(p58). Another phase II showed significant decreases in ALT levels and liver fat content
trial targeting the efficacy of PF-06865571 is currently ongoing (NCT02855164)(p67). Additionally, results from a phase II trial
and is projected to conclude in 2024 (NCT04321031). supported the continued development of another FXR agonist,
EDP 305–101 (NCT03421431)(p68). Finally, an FXR agonist called
AMP-activated protein kinase activators HEC96719 was measured for safety and tolerability in a phase I
AMP-activated protein kinase (AMPK) is an important kinase for trial, but the results were not posted (NCT04422496).
regulating energy homeostasis and maintaining physiological
cell activities. Activation of AMPK in the liver of diet-induced Single/dual/triple agonist of GLP1R/GCGR/GIPR
obese mice can inhibit DNL and enhance fatty acid oxidation, Glucagon-like peptide 1 (GLP1) and glucose-dependent insulino-
leading to decreased steatosis and inflammation(p59). Metformin tropic polypeptide (GIP) are both incretins. GLP1 can stimulate
functions as an indirect agonist of AMPK and has a strong track insulin secretion, whereas GIP stimulates insulin or glucagon
record for reducing hepatocyte glucose synthesis and improving depending on the level of blood glucose(p69). Several agonists of

www.drugdiscoverytoday.com 9
 KEYNOTE (GREEN) Drug Discovery Today d Volume 29, Number 3 d March 2024

TABLE 2
Combination therapy in NAFLD and NASH
Drug (class) Mechanism Cohort Phase NCT no. Refs
LYS006 (LTA4H inhibitors) + LJN452 (FXR Inhibits LTA4H; activates FXR; inhibits bile acid NAFLD II NCT04147195 NA
agonists) synthesis
Selonsertib (ASK1 inhibitors) + firsocostat Inhibits the formation of inflammatory cytokines; Bridging II NCT03449446 (p84)

(ACC inhibitors) + cilofexor (FXR agonists) reduces gene expression related to fibrosis; inhibits fibrosis or
excessive apoptosis; reduces liver fibrosis; reduces compensated
intrahepatic sinusoidal resistance cirrhosis
(p85)
Semaglutide (GLP1R agonists) + cilofexor Inhibits hepatic gluconeogenesis; activates both NASH II NCT03987074
(FXR agonists) + firsocostat (ACC inhibitors) GLP1R and GLP; reduces liver fibrosis; reduces
KEYNOTE (GREEN)

intrahepatic sinusoidal resistance; reduces DNL

Efinopegdutide (GLP1R Increases insulin secretion and sensitivity; inhibits the Pre-cirrhotic II NCT05877547 NA
agonists) + semaglutide (GLP1R agonists) release of glucagon; inhibits hepatic gluconeogenesis; NASH
activates both GLP1R and GLP
(p59)
PF-06865571 (DGAT1 and DGAT2 Inhibits DGAT2; inhibits DNL; improves hepatic NASH and F2/ II NCT04321031
inhibitors) + PF-05221304 (ACC inhibitors) steatosis 3
K-877-ER (PPAR agonists) + CSG452 (SGLT Activates PPARa; inhibits SGLT; reduces glucose NASH II NCT05327127 NA
inhibitors) content
Semaglutide (GLP1R agonists) + cilofexor Inhibits hepatic gluconeogenesis; activates both F4 II NCT04971785 NA
(FXR agonists) + firsocostat (ACC inhibitors) GLP1R and GLP; reduces liver fibrosis; reduces
intrahepatic sinusoidal resistance; reduces DNL

GLP1 receptor (GLP1R) and/or GIP receptor (GIPR) have received cirrhosis, indicating an association between T2DM and NAFLD.
approval for T2DM. Given the clinical characteristics that T2DM Sodium glucose co-transporter 2 (SGLT2), a subtype of the SGLT
and NAFLD share in common, medications aimed at glucose or family, is pivotal in reabsorbing glucose in kidneys. SGLT2 inhi-
lipid metabolism could be a promising and innovative treatment bitors (SGLT2i) promote weight loss in both non-diabetics and
option for NAFLD. people with T2DM by inhibiting glucose reuptake in kidney(p74).
The phase II clinical trial of semaglutide demonstrated In patients with T2DM and NAFLD, miR-34a-5p is overexpressed,
achievement of the primary endpoint: resolution of NASH with- but SGLT2is such as empagliflozin downregulate miR-34a-5p to
out worsening of fibrosis, compared with the placebo group impede HSC activation, and ultimately improve NAFLD-related
(NCT02970942)(p70). A phase III clinical trial of semaglutide is fibrosis(p75). Empagliflozin treatment in ApoE-/- mice resulted in
currently recruiting (NCT04822181). Cotadutide, a dual agonist decreased fasting glucose, cholesterol and TG levels, as well as
of GLP1R and glucagon receptor (GCGR), reduced lipid accumu- improvements in lipid metabolism, inflammation and ER
lation and furthermore improved liver inflammation and fibrosis stress(p76). A 24-week clinical trial found that the SGLT2i dapagli-
in patients with T2DM(p71). The results of a phase II trial of flozin reduced the levels of steatosis- and fibrosis-linked biomark-
efinopegdutide, a dual-target co-agonist of GLP1R and GCGR, ers such as ALT and glutamyl transferase (GTT), and it helped to
demonstrated a much greater reduction in liver fat content at improve steatosis and fibrosis(p77). A phase III trial aimed to assess
the 10 mg dose compared with the 1 mg dose group the safety and efficacy of dapagliflozin is expected to be com-
(NCT04944992)(p72), and a new phase I trial of efinopegdutide pleted by July 2024 (NCT03723252). Finally, canagliflozin exhib-
is recruiting patients (NCT06052566). Recently, efinopegdutide ited positive outcomes in terms of NAFLD in individuals with
was granted fast track designation from the FDA as a potential diabetes mellitus(p78). A phase IV study suggested that treatment
treatment for patients with NASH. with ipragliflozin led to improvements in fat reduction and hep-
Tirzepatide, a dual agonist of GLP1R and GIPR, significantly atic steatosis in individuals with both T2DM and NAFLD
reduced NASH-related biomarkers including ALT, AST and (NCT02875821)(p79).
keratin-18 in a clinical phase II test on patients with T2DM
(NCT03131687)(p73). Another phase II trial of tirzepatide is ongo- ATP citrate lyase inhibitors
ing (NCT04166773). Pemvidutide, a dual agonist of GLP1R and ATP citrate lyase (ACLY) is a vital enzyme involved in synthesiz-
GCGR, was assessed for safety and tolerability in patients with ing cholesterol and fatty acids. In one preclinical trial, pharmaco-
NAFLD in a phase I trial (NCT05006885). Survodutide is cur- logical inhibition of ACLY led to improvements in the
rently being assessed for its effectiveness in NASH in a phase II histological features of NASH and in levels of blood glucose,
trial (NCT04771273). Finally, a triple agonist of GCGR, GIPR TGs and related metabolic parameters(p80). Bempedoic acid, a
and GLP1R, retatrutide, showed positive outcomes in a phase II novel antihyperlipidemic drug, is under assessment for its effec-
substudy. The reduction of liver fat depended on the dosage, tiveness in liver fat reduction in patients with NAFLD
and NASH resolution was observed in a high percentage of (NCT06035874). However, only a few drugs have been designed
participants. specifically to target ACLY, and most have not yet advanced to
clinical trials. One new ACLY inhibitor is 326E, which demon-
Sodium glucose co-transporter 2 inhibitors strated excellent effects in combating hypercholesterolemia
Many patients with NAFLD have the comorbidity of T2DM or and atherosclerosis(p81). 326E could hold potential for treating
obesity. T2DM exacerbates NAFLD progression to NASH or even NASH. More evidence is needed to support the potential

10 www.drugdiscoverytoday.com
Drug Discovery Today d Volume 29, Number 3 d March 2024 KEYNOTE (GREEN)

application of ACLY inhibitors in NAFLD, and their safety and NGM282, an engineered FGF19 analog, was found to be safe in
tolerability also warrant further examination and consideration. both patients with compensated cirrhosis (NCT04210245) and
patients with NASH (NCT02443116). Additionally, BFKB8488,
Acetyl-CoA carboxylase inhibitors an agonist of both FGFR1 and Klothob, reduced TG levels and
Acetyl-CoA carboxylase (ACC) is an enzyme that limits the rate was tolerated in patients with T2DM or NAFLD
of DNL by catalyzing the formation of malonyl-CoA. There are (NCT03060538)(p86).
two isoforms of ACC: ACC1 and ACC2. The inhibition of ACC Liver X receptor (LXR) has both positive and negative impacts
resulted in reduced adipose synthesis and steatosis in vivo and on NAFLD and NASH. When activated, it is thought to be anti-
hindered stellate cell activation in vitro, leading to improvements inflammatory, to enhance insulin sensitivity and to aid in liver
in NAFLD and NASH(p82). GS-0976, a dual inhibitor of ACC1 and fibrosis regression and steatosis remission. When inhibited, it

KEYNOTE (GREEN)
ACC2, suppressed DNL and lowered biomarkers related to fibro- can prevent the occurrence of hyperlipidemia. Thus, whether
sis, but resulted in increased levels of serum TGs in a phase II trial to enhance or inhibit LXR activity for therapeutic purposes
(NCT02856555)(p83). Clesacostat, another ACC1 and ACC2 inhi- remains controversial(p87). Oltipraz is an LXRa inhibitor. A 24-
bitor, was found to reduce liver fat in a dose-dependent manner, week phase II clinical trial demonstrated that liver fat content
with a good safety and tolerability profile (NCT03248882). was reduced to a greater degree in the high-dose group
Because the inhibition of ACC results in increased expression (NCT01373554)(p88). Another two phase III trials
of SREBP1 and TG accumulation, and because inhibiting DGAT2 (NCT02068339, NCT04142749) testing the effectiveness and
can downregulate SREBP1 and genes associated with lipid syn- safety of oltipraz in patients with NAFLD (but without liver cir-
thesis, combining these two categories of drugs led to positive rhosis) have been completed, although no results have been
outcomes in a phase II clinical trial (NCT03776175). posted thus far.
Chemokines are polypeptides with the ability to recruit
Combination therapy monocytes, neutrophils and lymphocytes. C–C chemokine
The development of NASH involves multiple and intricate path- receptor 2 (CCR2) activation leads to inflammation and HSC
ways, and potentially pathway crosstalk, which combined repre- activation, whereas CCR5 activation promotes collagen accumu-
sent a substantial hurdle to the development of a single, lation and fibrosis release. Cenicriviroc is a dual antagonist of
universally efficacious medication. Thus, it might be challenging CCR2 and CCR5. In the phase III AURORA trial, cenicriviroc
to devise a sole treatment that is applicable to all individuals. did not show efficacy in improving liver fibrosis, but the drug
Instead, adopting a combinatory therapy approach that targets was well tolerated in patients with NASH and liver fibrosis
multiple factors simultaneously could be the optimal therapeutic (NCT03028740)(p89).
approach (Table 2). Other, less-well-studied therapeutic targets are discussed
One example is a phase II clinical trial that combines LYS006, below.
a leukotriene A4 hydrolase (LTA4H) inhibitor, with LJN452, an Amine oxidase copper-containing 3 (AOC3) has been found
FXR agonist. The trial suggested an improvement in fibrosis to promote hepatic steatosis progression. One inhibitor of
among the group treated with LYS006. However, it is unclear AOC3, BI 1467335, reduced ALT in a dose-dependent manner
whether LYS006 alone or in combination with LJN452 produced in patients with NASH (NCT03166735)(p90).
a similar increase in fibrosis (NCT04147195). A phase II clinical Pan-phosphodiesterase (pan-PDE) also serves as a potential
trial is evaluating the safety and efficacy of the DGAT2 inhibitor therapeutic target. ZSP1601, an inhibitor of pan-PDE, improved
PF-06865571 alone or in combination with the ACC inhibitor liver fat content and fibrosis, and was well tolerated in patients
PF-05221304. The primary endpoint is NASH resolution without with NAFLD (NCT04140123)(p91).
worsening of fibrosis or improvement in fibrosis by 1>/= or both A3 adenosine receptor (A3AR, also known as ADORA3) is
and the trial is set to conclude in 2024 (NCT04321031). majorly expressed in liver cells. Namodenoson, an A3AR agonist,
A phase IIb trial examined the safety and efficacy of selon- could protect liver from inflammation, and a phase II trial of
serib, firsocostat and cilofexor alone or in combination for the namodenoson is ongoing (NCT04697810). Additionally,
treatment of bridging fibrosis or compensated cirrhosis, with a FM101, an A3AR modulator, was evaluated for its safety and tol-
primary endpoint focused on one-stage fibrosis regression with erability in NASH therapy in a phase II trial (NCT04710524).
no worsening of NASH. The findings showed that the combina-
tions of firsocostat & cilofexor, cilofexor & selonserib, and firso-
costat & selonsertib (15.5%) performed better than firsocostat or Concluding remarks and future prospectives
cilofexor alone, but worse than selonsertib alone Currently, liver biopsy remains the gold standard for diagnosing
(NCT03449446)(p84). Another trial showed that the combination and staging NAFLD, but its use is limited owing to the high risk
of semaglutide, cilofexor and firsocostat might produce addi- of complications coupled with high costs and incompliance from
tional benefits compared with semaglutide monotherapy(p85). patients. Consequently, there is an urgent need to establish an
Other combinatory clinical trials (NCT05877547, early, reliable and non-invasive diagnostic approach. For exam-
NCT05327127, NCT04971785) are ongoing. ple, circulating free DNA detection could be a good option, as
it can be less invasive and more specific. NAFLD is normally asso-
Other approaches ciated with multiple extrahepatic complications, particularly in
Like FGF21, as discussed above, FGF19 acts as a regulator of lipid the cardiovascular system. Therefore, constructing a precise
and glucose homeostasis, thus influencing liver fibrogenesis. model that portrays the concurrent presence of multiple diseases

www.drugdiscoverytoday.com 11
 KEYNOTE (GREEN) Drug Discovery Today d Volume 29, Number 3 d March 2024

and associated clinical features is challenging but imperative. spectrum. Optimistically, more comprehensive models will be
Future research directions in NAFLD and NASH therapy might established in the near future, and the organoid model might
include the following. be a good option. One exemplary is a multicellular liver orga-
noid, which supports the investigation of NAFLD development
Seeking the discovery of novel anti-NAFLD/NASH medications in individuals infected by hepatitis C virus(p99). Moreover,
Few clinical successes have been achieved in NAFLD and NASH humanized mouse models could be promising. In a mouse model
therapy, despite the existence of over 1,500 registered clinical tri- with mouse hepatocytes replaced by human hepatocytes,
als (as of 21 January 2024). Due to the complex etiology of the improved liver fibrosis was observed when TAZ in human hepa-
disease, inherited factors and genetic variants in patients with tocytes was silenced(p100).
NAFLD and NASH, novel therapies are needed, such as RNA- In summary, some challenges exist when translating preclin-
KEYNOTE (GREEN)

based drugs. For example, a phase I trial of AZD2693 (targeting ical studies to the clinical arena. Deeper understanding of the
PNPLA3) is ongoing, (NCT04483947) and another phase I trial pathogenesis of NAFLD and NASH will produce more disease-
of AMG 609 (also targeting PNPLA3) has been completed, relevant targets. The combination of non-pharmacological inter-
although no results have been posted (NCT04857606). Further- ventions and pharmacological treatments, including therapies
more, the upregulated expression of HSD17B13 promotes the combining different categories of drugs targeting NAFLD and
development of NAFLD, and there are many trials targeting NASH, is expected to produce safer and more effective means
HSD17B13(p92). For example, ARO-HSD significantly decreased to curb these diseases.
levels of HSD17B13 and serum ALT in patients with NASH
(NCT04202354)(p93). GSK4532990 is being evaluated for its abil-
ity to improve histological fibrosis in patients with NASH and F3
Author contributions
fibrosis (NCT05583344), and a phase I trial will assess the safety S.X. conceptualizes this work; S.W., L.W. and P.C.E. wrote and
and tolerability of INI-822 in healthy individuals and patients edited the manuscript.
with NASH (NCT05945537). Additionally, AZD4076, a miRNA
targeting miR-23-27-24, is being assessed in healthy male indi- Conflict of interest statement
viduals in a phase I trial (NCT02612662). None.

Elucidating shared mechanisms driving CVD in NAFLD and

NASH conditions CRediT authorship contribution statement
Because of the association between CVD and liver disease and Shulin Wei: Writing – original draft. Li Wang: Writing –
the high mortality rate caused by CVD in patients with NAFLD, review & editing. Paul C. Evans: Writing – review & editing.
pharmacotherapies targeting CVD might be important for Suowen Xu: Supervision, Conceptualization.
NAFLD and NASH treatment(p94),(p95). For example, increased
levels of steatotic hepatocyte-derived small extracellular vesicles
Data availability
(sEVs), which inhibit the expression of ATP-binding cassette
No data was used for the research described in the article.
transporter A1, can lead to reduced cholesterol efflux and aggra-
vated atherosclerosis in NAFLD mice and patients(p96). Therefore,
it could be beneficial to reduce atherosclerosis by inhibiting the Acknowledgements
secretion of sEVs. Notably, EVs are membranous particles carry- This study was supported by grants from the National Key
ing cargo proteins or miRNA from donor cells to recipient cells, R&D Program of China (Grant No. 2021YFC2500500), the
suggesting that EVs can be designed as a drug delivery system. National Natural Science Foundation of China (Grant Nos.
For instance, incorporating CRIPSR-Cas9 ribonucleoprotein into 82370444 and 82070464) and the Strategic Priority Research Pro-
EVs could potentially achieve liver-targeted gene therapy(p97). gram of the Chinese Academy of Sciences (Grant No.
Other mechanisms leading to exaggerated CVD in patients with XDB38010100). This work was also supported by the Program
NAFLD include secreted proteins, metabolites/lipids and other for Innovative Research Team of The First Affiliated Hospital of
bloodborne factors. USTC (CXGG02), Anhui Provincial Key Research and Develop-
ment Program (Grant No. 202104j07020051) and Anhui Provin-
Biotechnology advances in studying NAFLD and NASH cial Natural Science Foundation (Grant No. 2208085J08). S.X. is a
Currently, numerous animal models are available for studying recipient of a Humboldt fellowship from the Alexander von
the etiology and therapeutics of NALFD and NASH, including Humboldt Foundation, Germany. We thank Peter J. Little
dietary and genetic models(p98). However, very few models are (University of Queensland, Australia) for critical reading, edits
able to fully reproduce the human disease pathogenesis and full and insightful discussions.

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