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Citation: Prikhodko, V.A.;
Bezborodkina, N.N.; Okovityi, S.V.
Pharmacotherapy for Non-Alcoholic
Fatty Liver Disease: Emerging
Targets and Drug Candidates.
Biomedicines 2022,10, 274.
https://doi.org/10.3390/
biomedicines10020274
Academic Editor: Henricus
A.M. Mutsaers
Received: 16 December 2021
Accepted: 24 January 2022
Published: 26 January 2022
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biomedicines
Review
Pharmacotherapy for Non-Alcoholic Fatty Liver Disease:
Emerging Targets and Drug Candidates
Veronika A. Prikhodko 1,* , Natalia N. Bezborodkina 2and Sergey V. Okovityi 1,3
1Department of Pharmacology and Clinical Pharmacology, Saint Petersburg State Chemical and
Pharmaceutical University, 14A Prof. Popov Str., 197022 St. Petersburg, Russia;
sergey.okovity@pharminnotech.com
2Zoological Institute, Russian Academy of Sciences, 1 Universitetskaya emb., 199034 St. Petersburg, Russia;
natalia.bezborodkina@zin.ru
3Scientific, Clinical and Educational Center of Gastroenterology and Hepatology, Saint Petersburg State
University, 7/9 Universitetskaya emb., 199034 St. Petersburg, Russia
*Correspondence: veronika.prihodko@pharminnotech.com
Abstract:
Non-alcoholic fatty liver disease (NAFLD), or metabolic (dysfunction)-associated fatty liver
disease (MAFLD), is characterized by high global incidence and prevalence, a tight association with
common metabolic comorbidities, and a substantial risk of progression and associated mortality.
Despite the increasingly high medical and socioeconomic burden of NAFLD, the lack of approved
pharmacotherapy regimens remains an unsolved issue. In this paper, we aimed to provide an update
on the rapidly changing therapeutic landscape and highlight the major novel approaches to the
treatment of this disease. In addition to describing the biomolecules and pathways identified as
upcoming pharmacological targets for NAFLD, we reviewed the current status of drug discovery
and development pipeline with a special focus on recent evidence from clinical trials.
Keywords:
non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; chronic liver disease;
hepatoprotection; metabolic disorders
1. Introduction
Non-alcoholic fatty liver disease (NAFLD) includes a range of chronic conditions
characterized by excessive hepatic lipid accumulation, defined by the presence of steatosis
in >5% of hepatocytes, in the absence of significant alcohol consumption or other causes
of liver injury [
1
]. The global prevalence of NAFLD is currently estimated at 25% [
2
],
and is projected to increase by 21% by 2030 [
3
]. About 20–25% of NAFLD cases are
classified as non-alcoholic steatohepatitis (NASH), which has a substantially higher risk
of progression to liver fibrosis, cirrhosis, and end-stage liver disease, and hepatocellular
carcinoma (HCC) [
3
]. Considering the highly heterogeneous pathogenesis of metabolic
liver diseases, the term ‘metabolic (dysfunction)-associated fatty liver disease’ (MAFLD)
has recently been proposed as a broader alternative to the conventional ‘NAFLD’ [4].
Due to the increasingly high medical and socioeconomic burden of this disease as
well as its strong association with obesity, metabolic and cardiovascular disorders, NAFLD
pharmacotherapy has been the focus of latest research [
3
,
5
]. However, to date, no drug has
been approved by neither the European Medicines Agency nor the United States Food and
Drug Administration (FDA) for NAFLD [2,6].
In the present paper, we aimed to review the most recent data on major pharmaco-
logical targets in this disease, summarize the clinical evidence for novel investigational
agents as well as currently marketed drugs, and highlight the latest advances in the drug
development pipeline for NAFLD.
Biomedicines 2022,10, 274. https://doi.org/10.3390/biomedicines10020274 https://www.mdpi.com/journal/biomedicines
Biomedicines 2022,10, 274 2 of 34
2. THRβAgonists
Thyroid hormone receptor beta (THR
β
) is a nuclear receptor and a transcription factor
that mediates the genomic effects of thyroid hormones. THR
β
1 is the predominant isoform
expressed in the human liver, while other THRs are also found in the heart, brain, kidney,
skeletal muscle, and other tissues. Upon activation by triiodothyronine or other agonist,
THR
β
displaces corepressor proteins from the deoxyribonucleic acid (DNA) and facilitates
coactivator binding, thus allowing for gene transcription. THR
β
can form heterodimers
with other THR or nuclear receptor superfamily proteins, such as liver X receptor or
peroxisome proliferator-activated receptors (PPAR) [7].
Hepatic THR
β
upregulate free fatty acid (FA) uptake and oxidation, lipophagy and
lipolysis, and promote mitochondrial biogenesis and respiration, leading to increased
adenosine triphosphate (ATP) consumption and energy expenditure. THR
β
have also been
shown to induce transcriptional activation of bile acid (BA) synthesis, biliary lipid secretion,
and cholesterol serum clearance, leading to a decrease in proatherogenic lipoprotein lev-
els [
8
,
9
]. Additionally, THR
β
stimulates normal hepatocyte proliferation, at the same time
downregulating nuclear signaling pathways and inhibiting tumour growth and metastasis
formation in HCC [10].
Selective THR
β
agonists that are currently being developed for the treatment of
NAFLD include resmetirom (MGL-3196), VK2809 (MB 07811), TERN501, ASC41, and
MGL-3745
. In a 36-week phase 2 randomized clinical trial (RCT) with an additional
36-week
open label extension, resmetirom was safe, well tolerated, and significantly im-
proved lipid profiles, liver steatosis (as indicated by magnetic resonance imaging/proton
density fat fraction (MRI-PDFF)), liver stiffness (assessed by transient elastography), and
N-terminal type III collagen pro-peptide (Pro-C3) levels in patients with biopsy-confirmed
non-alcoholic steatohepatitis (NASH) [
11
], supporting its further evaluation in three ongo-
ing phase 3 trials (NCT04197479, NCT04951219, NCT03900429). The lipid-lowering effect
of resmetirom was accompanied by a significant improvement in alanine aminotransferase
(ALT) and
γ
-glutamyl transpeptidase (GGT) levels as well as a reduction in NAFLD activity
(NAS) and enhanced liver fibrosis (ELF) scores [11].
VK2809 (MB07811) is a prodrug yielding an active metabolite via oxidation by hepatic
CYP3A4 enzyme. A 12-week phase 2a RCT found VK2809 to possess significant antisteatotic
activity in NAFLD patients [
12
]. ASC41, also a prodrug, improved serum lipids and liver
histology in rats [
13
], and subsequently demonstrated a good safety profile and substantial
hypolipidemic activity in healthy volunteers [
14
], advancing to phase 2 (NCT05118360).
A newer THRβagonist, TERN-501, reduced serum cholesterol levels and attenuated liver
steatosis and fibrosis in rodent models of hyperlipidaemia and NASH [
15
], and has been
recently approved for phase 1 clinical trials [
16
,
17
]. MGL-3745 is being evaluated in
preclinical studies, and no data regarding its therapeutic activity are available yet. Two
fixed-dose combinations (FDC), ASC43 and ASC45, containing ASC41 and either a FA
synthase (FASN) inhibitor (ASC40) or a farnesoid X receptor (FXR) agonist (ASC42), are
being evaluated in preclinical and phase 1 clinical trials, respectively [16].
3. Lipogenesis Inhibitors
Acetyl-CoA carboxylase (ACC) is a multi-subunit enzymatic complex that catalyzes
the irreversible carboxylation of acetyl-coenzyme A (CoA) to produce malonyl-CoA. Acetyl-
and malonyl-CoA are then utilized by FASN to synthesize palmitate, which is converted
to stearate by the elongation of long-chain fatty acids family member 6. Stearoyl-CoA
desaturase 1 (SCD1) converts stearoyl-CoA and palmitoyl-CoA into unsaturated fatty
acids, which are later esterified with glycerol by several transferases, including diglyceride
acyltransferase (DGAT), to produce triacylglycerides (TAG). Two DGAT isozymes have
been identified so far: DGAT1 and DGAT2, which appear to be primarily responsible for
esterifying exogenous and endogenous fatty acids, respectively [
18
]. ACC, FASN, SCD1,
and DGAT catalyze the rate-limiting steps in the de novo lipogenesis, and have therefore
been considered suitable targets for the treatment of NAFLD.
Biomedicines 2022,10, 274 3 of 34
However, some animal evidence suggests that inhibition of TAG synthesis may worsen
hepatic inflammation and fibrosis [
19
], and alter intestinal barrier function, leading to diar-
rhoea and steatorrhoea [
20
]. These findings might represent a challenge for the development
of lipogenesis inhibitors and potentially limit their clinical application to early stages of
liver steatosis not associated with inflammation and fibrogenesis [21].
3.1. ACC Inhibitors
Firsocostat (GS-0976) and clesacostat (PF-05221304) are selective ACC inhibitors cur-
rently under development for combination therapy of NASH. Since ACC is indirectly
inhibited by FXR [
22
], firsocostat has been combined with the FXR agonist (see 4.1 FXR
agonists) cilofexor in order to maximize the resulting effects. In the phase 2b ATLAS trial,
20 mg/d firsocostat + 30 mg/d cilofexor provided significant reductions in NAS scores,
liver steatosis, lobular inflammation and hepatocellular ballooning (HCB), and improved
liver biochemistry over 48 weeks in patients with septal fibrosis due to NASH [
23
]. Cle-
sacostat (2–50 mg/d) has demonstrated good efficacy in terms of reducing liver steatosis,
and is being developed in combination with the DGAT2 inhibitor ervogastat to address the
frequently observed elevation of serum TAG, a known effect of ACC inhibitors [
24
]. Two
phase 2 studies are ongoing to determine the optimal doses of both agents for NASH with
and without liver fibrosis (NCT04399538, NCT04321031).
3.2. FASN Inhibitors
Known investigational FASN inhibitors include ASC40 (TVB-2640), FT-8225, and
ASC44F, a fixed-dose combination containing ASC40 and ASC42 (a FXR agonist; see
FXR agonists). ASC40 (50–150 mg/d) provided near complete (~90%) inhibition of de
novo lipogenesis and reduced liver steatosis in obese subjects with substantial NAFLD
risk [
25
]. In the phase 2 FASCINATE-1 trial, ASC40 at lower doses (25 or 50 mg/d)
improved ALT and low-density lipoprotein (LDL) cholesterol levels, attenuated liver
steatosis, fibrosis, lipotoxicity, dyslipidemia, and hepatic insulin resistance in obese NASH
patients [
26
]. A subsequent phase 2 study, FASCINATE-2, has been initiated (NCT04906421).
The anti-inflammatory and antifibrotic properties of ASC40 have been linked to inhibited
proinflammatory cytokine production, T-cell differentiation, and repression of collagen
synthesis [
26
]. FT-8225 and ASC44F have entered preclinical development [
16
,
27
]; no data
regarding their efficacy is available yet.
3.3. SCD1 Inhibitors
Aramchol (C20-FABAC) is a synthetic conjugate of arachidic and cholic acids and the
first-in-class inhibitor of SCD1. In addition to inhibiting de novo lipogenesis and hepatic
stellate cell (HSC)-mediated fibrogenesis [
28
], aramchol is capable of reducing serum choles-
terol levels and promoting gallstone dissolution via stimulation of macrophage cholesterol
efflux and solubilization of cholesterol [
29
]. In the recently completed
52-week
phase 2b
ARREST trial, aramchol (600 mg/d) did not meet the primary endpoint (a significant
decrease in hepatic TAG), but was well tolerated and improved liver histology and ALT
levels [
30
]. The phase 3 ARMOR RCT has been designed to evaluate the safety and effi-
cacy of 300 mg/d aramchol in patients with biopsy-confirmed liver fibrosis due to NASH
(NCT04104321).
3.4. DGAT Inhibitors
Ervogastat (PF-06865571), a selective DGAT2 inhibitor, significantly reduced liver fat
fraction in patients with mild NAFLD [
31
]. Two phase 2 RCT are underway to assess
the safety and efficacy of ervogastat alone and in combination with clesacostat in NASH
patients with and without liver fibrosis (NCT04399538, NCT04321031). ION224 (IONIS-
DGAT2
Rx
) is a ligand-conjugated chimeric antisense oligonucleotide designed to suppress
the biosynthesis of DGAT2. ION224 reduced total liver fat content and several fibrosis
biomarker levels in a small-scale trial in patients with type 2 diabetes mellitus (T2DM) and
Biomedicines 2022,10, 274 4 of 34
NAFLD [
32
], and is planned to be evaluated in a longer phase 2 RCT in nondiabetic subjects
(NCT04932512). Newest DGAT2 inhibitors include PF-07202954, and DGAT1 inhibitors,
VK1430, SNP-610, and SNP-630; the latter two molecules are reported to have additional
CYP2E1-inhibiting properties [33].
3.5. ω-3 PUFAs
ω
-3 Polyunsaturated fatty acids (
ω
-3 PUFA) are long-chain FA characterized by the
presence of a double bond three atoms away from the terminal methyl group in their
chemical structure. The three most common biologically active
ω
-3 PUFA include
α
-
linolenic (ALA) acid and its metabolites eicosapentaenoic (EPA) and docosahexaenoic
(DHA) acids. ω-3 PUFAs cause transcriptional repression of the key enzymes involved in
hepatic glycolysis and de novo lipogenesis, such as ACC, FASN, and L-pyruvate kinase [
34
].
Increased
ω
-3 PUFA intake results in their increased incorporation into cell mem-
brane phospholipids, corresponding to a positive shift in the
ω
-3/
ω
-6 ratio. This leads
to decreased availability of the
ω
-6 arachidonic acid (AA), and the subsequent substrate-
dependent inhibition of eicosanoid inflammatory mediator production by the leukocytes.
Moreover, EPA and DHA have been found to suppress leukocyte chemotaxis and adhesion
molecule expression, altogether providing a multimodal anti-inflammatory effect [
35
]. In
addition,
ω
-3 PUFA are also known for their prominent antioxidant, regenerative, and
antitumour properties [36].
A 6-week treatment with
ω
-3 PUFA (64% ALA + 21% EPA + 16% DHA) was associ-
ated with significant improvement of hepatic proteomic and plasma lipidomic markers of
lipogenesis, lipotoxicity, oxidative stress, and mitochondrial respiration in patients with
biopsy-confirmed NASH [
37
]. The increase in plasma ALA and DHA levels, and the subse-
quent decrease in AA levels correlated with the percentage of patients with improvements
in NAS scores, lobular inflammation, and HCB. However, the overall liver histology and
body weight were not significantly altered by ω-3 PUFA treatment [38].
A systematic review and a meta-analysis confirmed that DHA and EPA attenuate liver
steatosis with no significant weight loss in adults [
39
], and a small-scale RCT reported
the same effects for dietary DHA supplementation in children [
40
]. More recently, three
meta-analyses, including up to 22 RCT and more than 1300 patients, found
ω
-3 PUFA to
significantly decrease ALT, aspartate aminotransferase (AST), GGT levels, liver fat content
and insulin resistance, having no significant effect on body weight in NAFLD [41–43].
Epeleuton (15-hydroxyeicosapentaenoic acid ethyl ester, DS102) is a second-generation
synthetic EPA derivative. In a 16-week phase 2a RCT in obese subjects with NAFLD,
epeleuton (2 g/d) improved circulating inflammatory markers, lipid profiles, and insulin
resistance, but failed to reach either of the primary endpoints including reductions in ALT
levels and liver stiffness [
44
]. Since a slight dose-dependent attenuation of liver steatosis
was observed, further trials of longer duration are planned [45].
Icosabutate (NST-4016) is a structurally engineered
ω
-3 PUFA ether characterized to
increased liver exposure due to direct absorption into the portal vein [
46
]. The 62-week
phase 2b ICONA trial is underway to evaluate the efficacy of icosabutate (300 or 600 mg/d)
in patients with biopsy-confirmed NASH (NCT04052516). Interim analysis data indicated
significant dose-dependent decreases among both dosage groups in ALT, AST, GGT, and
alkaline phosphatase (ALP) levels, while patients dosed with 600 mg/d icosabutate also
had improvements in non-invasive fibrosis and inflammatory biomarker profiles [46].
4. Bile Acid Metabolism Modulators
4.1. FXR Agonists
Farnesoid X receptor (FXR), also known as bile acid receptor, is a nuclear receptor
expressed at high levels in the liver and ileum. FXR acts as a BA sensor and governs a
major negative feedback loop in the BA, glucose, cholesterol, and TAG metabolism [
22
].
Many of the effects of FXR are directly mediated by fibroblast growth factors (FGF) 19 and
21, downstream messengers whose functions are briefly described in Fibroblast growth
Biomedicines 2022,10, 274 5 of 34
factor analogues. Upon increased postprandial release of BA into the intestine, activated
FXR induces the transcriptional repression of the rate-limiting enzyme cholesterol 7
α
-
monooxygenase (CYP7A1) and several transporters involved in BA biosynthesis and liver
uptake. CYP7A1 inhibition by FXR enhances the excretion of excessive cholesterol via the
canalicular transporters into bile as well as directly into the intestinal lumen. FXR also
upregulates the expression of bile salt export pump, multidrug resistance protein-3, and
organic solute transporter
α
/
β
, which facilitate BA efflux from hepatocytes and maintain
the enterohepatic circulation of bile [
47
]. FXR activation also favors bile acid conjugation
and detoxification, and stimulates biliary phospholipid excretion.
FXR and its downstream targets repress sterol regulatory element-binding protein 1
SREBP-1c, the major transcription factor for lipogenic pathways, thereby inhibiting FASN,
ACC, and SCD1. In addition, prandial glucose can increase the intestinal FXR activity via
post-translational modification, shifting the equilibrium towards glycogen deposition and
reducing blood glucose levels [22]. The multifaceted nature of FXR has made it one of the
most attractive novel targets for NAFLD therapy. Current FXR-activating drug candidates
include the sterol derivatives obeticholic acid (OCA), EDP-305, INT-767, and INT-787, and
the non-steroidal compounds MET409, tropifexor, cilofexor, vonafexor, TERN-101, ASC42,
EDP-297, HPG1860, and HPG7233.
OCA (Ocaliva
®
), the first-in-class FXR agonist, is approved by the FDA for non-
cirrhotic primary biliary cholangitis (PBC) and is nearing approval for liver fibrosis due to
NASH [
48
]. In phase 2 studies, OCA increased insulin sensitivity and reduced markers of
liver damage in patients with T2DM and NAFLD [
49
], and reduced ALT levels, improved
NAS scores, prevented and partially reversed fibrogenesis in non-diabetic, pre-cirrhotic
NASH patients [
50
]. Currently, the efficacy and safety of OCA are being evaluated in two
phase 3 trials, the REGENERATE trial in NASH/fibrosis patients (NCT02548351), and the
REVERSE trial in patients with compensated cirrhosis due to NASH (NCT03439254).
OCA and especially its taurine and glycine conjugates are known to actively bind
Takeda G-protein receptor 5 (TGR5), a cell membrane G protein-coupled receptor (GPCR)
that has been largely implicated in BA-associated pruritus development [
51
] and gallstone
formation [
52
]. Hence, several newer molecules, e.g., EDP-305 and INT-787, have been
structurally optimized in an attempt to avoid TGR5 engagement and potential safety
concerns [
53
]. However, PBC trials have shown that highly selective FXR agonists retain
their adverse effects, at the same time losing in overall efficacy [
54
,
55
]. EDP-305 was later
repurposed for use in NASH, and, along with its close analogue EDP-297, is now intended
to be reserved for future combination regimens following mixed interim results of the phase
2b ARGON-2 study [
56
]. Since FXR and TGR5 seem to exert additive metabolic effects,
dual FXR/TGR5 agonists with balanced activity towards both targets, such as INT-767 and
BAR502, have been proposed for further development for NAFLD treatment [57,58].
MET409, a structurally optimized fexaramine-derived FXR agonist with greater affinity
towards intestinal rather than hepatic FXR, is characterized by improved efficacy and a dif-
ferentiated adverse effect (pruritus and increased LDL-cholesterol levels) profile compared
to OCA [
59
], and is currently being evaluated in a phase 2a RCT alone and in combination
with empagliflozin (NCT04702490).
Tropifexor (LJN452), cilofexor (GS-9674), and vonafexor (EYP001) represent a group
of non-steroidal small molecules with structures different from both OCA and MET409,
resulting in a differential pattern of FXR-related gene expression [
60
] and possibly improved
anticholestatic activity. The parent compound for this group, turofexorate (WAY-362450),
completed a phase 1 study, but its development was discontinued thereafter [
61
]. Tropifexor
(200, but not 140 mcg/d) reduced ALT, GGT levels, body weight, and liver fat content,
and attenuated liver fibrosis in patients with biopsy-confirmed NASH in the 48-week
phase 2 FLIGHT-FXR RCT [
62
,
63
]. Two additional phase 2 trials are currently underway to
investigate the efficacy of tropifexor combinations with licogliflozin (NCT04065841) and
LYS006 (NCT04147195) in NASH/fibrosis and NAFLD/NASH, respectively.
Biomedicines 2022,10, 274 6 of 34
Cilofexor reduced liver fat content (100 mg/d) and GGT, N-terminal type IV collagen
pro-peptide, and serum primary BA levels (30 or 100 mg/d) over 24 weeks, but did not
affect liver elasticity and ELF scores in NASH patients [
64
]. A phase 2 RCT has been initiated
to evaluate the efficacy of a fixed-dosed combination of cilofexor and firsocostat, alone or
in combination with semaglutide, for compensated cirrhosis due to NASH (NCT04971785).
Vonafexor (100 mg/d for 12 weeks), a 2nd generation, highly selective non-steroidal FXR
agonist, has recently been found to improve liver steatosis, the fibro-inflammation marker
cT1 levels, and estimated glomerular filtration rate (eGFR) in NASH patients with normal
or mildly decreased eGFR [
65
]. Nidufexor (LMB763), a non-steroidal partial FXR agonist,
was being developed for NASH, liver fibrosis, and cholestatic liver disease, but appears
to have been discontinued despite seemingly promising results from a phase 2 study in
NASH subjects [66].
TERN-101 demonstrated a favorable safety profile and improved ALT levels and liver
steatosis in the phase 2a LIFT RCT in patients with pre-cirrhotic NASH [
67
]. ASC42
treatment was associated with biochemical and histological improvements in animal
NASH models [
16
], while no data are available yet for novel compounds HPG1860 and
HPG7233 [68].
4.2. FGF Analogues
Fibroblast growth factors (FGF), including FGF19 and FGF21, are a family of hormone-
like peptides with broad metabolic, transcriptional, and mitogenic activity. FGF19 is
released by the ileal enterocytes into the enterohepatic circulation in response to postpran-
dial FXR activation by BA. FGF21 is highly expressed in the liver and is released in response
to PPAR
α
, carbohydrate-response element-binding protein (ChREBP), and general control
nonderepressible 2 kinase activation in the presence high serum glucose, high FFA and low
amino acid levels [69]. FGF19 and FGF21 exert their physiological effects via activation of
transmembrane complexes of the enzyme
β
-klotho (KLB) and its FGF co-receptors (FGFR)
FGFR1c/2c/3c/4. FGF21 has the highest affinity for the FGFR1c/KLB complex, which is
expressed in the adipose tissue and central nervous system, while FGF19 primarily targets
FGFR4/KLB, which is found in hepatocytes [69].
Thus, FGF19 and FGF21 act as major downstream messengers in the FXR and PPAR
α
signaling, and control the negative feedback loops to inhibit BA synthesis and lipolysis,
respectively [
69
]. FGF19 represses CYP7A1, controls postprandial BA release into the in-
testinal lumen, inhibits hepatic gluconeogenesis, promotes glycogen deposition, and plays
an important role in the regulation of hepatocyte proliferation and tumorigenesis. FGF21
lowers the preference for glucose intake, modulates energy expenditure, and promotes
glucose and lipid uptake in the adipose tissue, which prevents ectopic lipid accumulation
in liver and skeletal muscle [69].
The investigational FGF19 analogue aldafermin (NGM-282) (0.3–3 mg/d) failed to
meet the primary endpoint of the 24-week phase 2b ALPINE 2/3 trial in NASH patients
with stage 2 or 3 liver fibrosis, defined as a
≥
1 NASH Clinical Research Network stage
improvement in fibrosis with no worsening of NASH. However, the drug was well tolerated
and was significantly superior to placebo in terms of NASH resolution, reduction of liver
steatosis and non-invasive markers of liver injury [
70
]. The ALPINE 4 trial is ongoing to
determine whether aldafermin could improve liver fibrosis and/or NASH in subjects with
compensated cirrhosis (NCT04210245).
Current FGF21 analogues include efruxifermin (AKR-001), BIO89-100, NN9500, BFKB8488A,
MK-3655, and GLP-1-Fc-FGF21 D1. Efruxifermin is a fusion protein of human immunoglob-
ulin G1 (IgG1) Fc domain linked to a modified human FGF21 with balanced affinity
towards FGFR1c/2c/3c. Efruxifermin (28–70 mg/d) improved lipoprotein profiles and
glycaemic control in T2DM patients, significantly attenuated liver steatosis in the 16-week
phase 2a BALANCED study [71], and is now being evaluated in three more phase 2 RCTs
(NCT05039450, NCT04767529, NCT03976401). BIO89-100 is a specifically engineered gly-
colpolyethylene glycol (PEG)-ylated FGF21 analogue that led to clinically meaningful
Biomedicines 2022,10, 274 7 of 34
reductions in liver fat content, markers of inflammation and fibrosis in a phase 1b/2a
proof-of-concept study in NASH patients [
72
]. Recently, the phase 2b ENLIVEN trial of
BIO89-100 for stage 2/3 fibrosis due to NASH has been initiated (NCT04929483), while two
phase 1 open-label studies are still underway (NCT05022693, NCT04048135). No preclinical
data is available yet for the investigational compound NN9500 [73].
BFKB8488A and MK-3655 are humanized bispecific anti-FGFR1c/KLB agonist mon-
oclonal antibodies (mAB). BFKB8488A was safe and adequately tolerated, reduced liver
steatosis in a dose-dependent fashion, and improved markers of cardiometabolic and liver
health in obese T2DM patients with NAFLD [
74
]. The phase 2b BANFF trial has been
initiated to explore the efficacy and safety of this compound in non-alcoholic liver fibrosis
(NCT04171765). MK-3655 (NMG313), an insulin-sensitizing anti-FGFR1c/KLB agonist
mAB, was effective against liver steatosis in obese and insulin-resistant NAFLD patients,
and has proceeded into a phase 2b study in pre-cirrhotic NASH with or without T2DM
(NCT04583423).
GLP-1-Fc-FGF21 D1 is a novel fusion protein incorporating a KLB-binding FGF21
variant and a glucagon-like peptide 1 receptor (GLP1R) agonist. In murine models of
T2DM and obesity, GLP-1-Fc-FGF21 D1 improved liver function, serum and hepatic lipid
profiles, and reduced body weight and NAS scores with an efficacy superior to either FGF21
or GLP1R agonists alone [
75
]. The PEGylated human recombinant FGF21 pegbelfermin
(ARX-618) has recently demonstrated suboptimal efficacy in compensated cirrhosis due
to NASH in the FALCON 2 trial, resulting in pending discontinuation and/or possible
repurposing for other indications [76].
5. Fibrogenesis Inhibitors
5.1. Galectin Antagonists
Galectins, formerly known as S-type lectins, are a family of carbohydrate-binding
proteins that are selective towards β-galactoside-containing glycans. To date, 15 subtypes
of galectins family have been identified in humans, of which galectin-1, -3, and -9 are
the most implicated in liver disease. Numerous experimental and clinical study results
suggest that galectins play important and diverse roles in fibrogenesis, cellular immunity
and inflammatory response, cell cycle regulation, apoptosis, regeneration, and tumorigene-
sis [77,78].
Galectin-1 promotes the proliferation, migration and activation of HSC, and the subse-
quent fibrogenesis via stimulation of transforming growth factor
β
(TGF
β
)/platelet-derived
growth factor signaling and disruption of cell adhesion. In HCC, galectin-1 promotes the
epithelial–mesenchymal transition, cell adhesion, metastasis, and immunosuppression.
However, elevated galectin-1 expression has also been found beneficial for liver regenera-
tion, liver allograft survival, and recovery after hepatic ischemia-reperfusion injury [77].
Galectin-3 has been identified as a pivotal regulator in the progression of hepatitis,
hepatic fibrosis, cirrhosis, and HCC. It was found to promote autocrine and paracrine HSC
activation and phagocytosis, mediate the TGF
β
-dependent fibrogenesis, and upregulate
the expression of certain profibrogenic cytokines such as interleukin (IL) 33. The exact role
of galectin-3 for liver cirrhosis is not as clear, but its increased expression has been linked
to accelerated cirrhosis development and deterioration of liver function [
77
]. However,
galectin-3 might be protective against adipose tissue inflammation, diabetes, and atheroscle-
rosis progression, most probably due to and its ability to scavenge the proinflammatory
and proapoptotic advanced glycation end products, the increased activation of the NF-
κ
B
signaling pathway, and the downregulation of NLRP3 inflammasome and IL1
β
expression
in immune cells [77].
A systematic review and meta-analysis by An et al. found that serum
galectin-3/9 levels
correlate with the risk of liver failure and cirrhosis, and high galectin-1/3 expression is associ-
ated with poorprognosis in HCC. However, the evidence for galectin involvement in chronic
liver disease remains controversial, and the impact of galectin-3 levels on NAFLD/NASH is
thought to be dependent upon the stage and severity of liver damage [
78
]. Hence, modern
Biomedicines 2022,10, 274 8 of 34
galectin-targeting drug candidates are intended for use in advanced NASH complicated by
liver fibrosis and/or cirrhosis.
GM-CT-01 and belapectin (GR-MD-02) are semi-synthetic polysaccharides (galac-
tomannan and galactoarabino-rhamnogalacturonan, respectively) having high affinity
towards extracellular galectin-3 and, to a lesser extent, galectin-1. Both compounds pro-
moted resolution of portal inflammation, HCB, liver fibrosis, and cirrhosis, and reduced
portal pressure in a toxin-induced liver injury model in rats [
79
]; however, only belapectin
seems to have advanced further into clinical development. In the 52-week phase 2b NASH-
CX study in subjects with NASH, liver cirrhosis, and portal hypertension, belapectin
(2 mg/kg/2 weeks) did not affect fibrosis or NAFLD activity, but reduced hepatic ve-
nous pressure gradient values and prevented the development of esophageal varices in a
sub-group of patients [
80
]. The phase 2/3 NAVIGATE trial has been initiated to further
evaluate belapectin in patients with liver cirrhosis due to NASH and clinical signs of portal
hypertension but without esophageal varices at baseline (NCT04365868).
GB1211, an oral small-molecule selective galectin-3 antagonist, was effective in several
preclinical fibrosis models, and well tolerated in human volunteers. A phase 1/2a trial
in NASH/fibrosis patients was approved but subsequently placed on hold due to an
undisclosed change in the clinical development strategy for this compound (NCT04607655).
5.2. TLR4 Antagonists
Toll-like receptor 4 (TLR4) belongs to the family of pattern recognition receptors that
activate the innate immune system by recognizing their major ligand, lipopolysaccharide
(LPS). In the liver, TLR4 are expressed in both parenchymal and non-parenchymal type
cells. TLR4 activation results in the activation of NF-
κ
B, mitogen-activated protein kinase
and interferon regulatory factor-mediated pathways, and the subsequent inflammatory
cytokine and interferon production [81].
TLR4 stimulate adhesion molecule expression and chemokine secretion by the HSC,
which induces Kupffer cell migration and the recruitment of extrahepatic monocytes into
the liver. TLR4 also downregulate HSC expression of the Bambi protein, an endogenous
TGF
β
receptor inhibitor, thus promoting profibrogenic TGF
β
signaling. Additionally, TLR4
can inhibit miR-29 expression and increase fibronectin production in the HSC, further
enhancing HSC activation and migration [82].
JKB-122 treatment (5 or 35 mg/d for 12 weeks) was well tolerated and significantly
improved ALT and AST levels, liver steatosis, and serum lipid profiles in a phase 2 study
in patients with NAFLD [
83
]. Another phase 2 RCT is planned to investigate whether
JKB-122 could ameliorate liver fibrosis due to NASH (NCT04255069). Recently, eritoran, a
synthetic bacterial lipid analogue, was found to significantly reduce ALT levels, lobular
inflammation, intrahepatic neutrophil infiltration, and liver fibrosis, but not liver steatosis,
in murine models of acute and chronic liver injury [81].
5.3. LOXL2 Inhibitors
Lysyl oxidase-like protein (LOXL) 2 is a histone modifier amine oxidase that has been
identified as the primary enzyme facilitating covalent crosslinking of collagen and elastin
fibers, thereby promoting collagen network formation and progression of liver fibrosis.
LOXL2 inhibition following the onset of fibrosis has been demonstrated to augment and
accelerate collagen degradation in rodent models [
84
]. Only a few studies have concerned
the role of LOXL3, a less common isozyme of LOXL, in fibrotic diseases, and its value as a
therapeutic target is somewhat controversial [85].
Simtuzumab (GS-6624), a humanized antagonist mAB, was one of the first LOXL2-
targeting drug candidates, that was discontinued after showing a lack of efficacy regarding
liver fibrosis and/or portal hypertension in two phase 2b trials in patients with bridging
fibrosis or compensated cirrhosis associated with NASH [86].
Novel LOXL2 antagonists are represented by orally available small molecules. PXS-
5153A, a dual LOXL2/LOXL3 inhibitor, reduced disease severity and improved liver
Biomedicines 2022,10, 274 9 of 34
function by diminishing collagen content and collagen crosslinks in two rodent models
of liver fibrosis [
87
]. Its cognate PXS-5382A, selective towards LOXL2, showed a satis-
factory pharmacokinetic profile in healthy volunteers (NCT04183517), and is expected to
advance into phase 2 trials. Another LOXL2 inhibitor, GB2064, was initially researched
for myelofibrosis, but appears to have had its potential indication list expanded to include
other fibrotic diseases [88].
5.4. ATX Inhibitors
Autotaxin (ATX) is a glycoprotein enzyme that converts membrane-derived lysophos-
pholipids into lysophosphatidic acid (LPA). LPA, in turn, acts as a multimodal signaling
molecule and causes cytoskeleton remodelling, alters cell proliferation and migration, and
promotes fibrogenesis as well as inflammatory reactions [
89
]. Non-competitive inhibition
of autotaxin/LPA signaling by the indole derivative PAT-505 resulted in a significant at-
tenuation of liver fibrosis in mouse models of NASH [
90
]. In addition, ATX induction has
been implicated in BA-mediated pruritus development, suggesting potentially favorable
safety profiles of ATX inhibitors. Two small-molecule ATX inhibitors, TJC0265 and TJC0316,
have been identified as lead compounds and are currently undergoing optimization and
preliminary in vivo testing [91].
6. Glucose Metabolism Modulators
6.1. PPAR Agonists
PPAR are a family of nuclear receptors that function as transcription factors and
play a regulatory role in glucose homeostasis, lipid metabolism, inflammatory response,
cell development and differentiation [
92
]. Up to now, three PPAR subtypes have been
identified: (1) PPAR
α
, expressed in the liver, adipose tissue, skeletal muscle, heart, and
kidney; (2) PPAR
γ
, expressed in the adipose tissue, colon, macrophages, pancreas, skeletal
muscle, etc.; and (3) PPAR
δ
(
β
), found ubiquitously. Upon ligand binding, PPAR induce
the release of corepressors and recruitment of coactivators, allowing for gene transcription.
Moreover, PPAR can regulate the mitogen-activated protein kinase pathways, and inhibit
inflammatory reactions via transrepression of several proinflammatory transcription factors
such as NF-κB [92].
Hepatic PPAR
α
stimulate mitochondrial FA uptake,
β
-oxidation, ATP production,
and ketogenesis. They upregulate glucose-sensing transcription factors ChREBP and sterol
regulatory element-binding transcription factor 1, and promote FGF21 expression, which
increases tissue insulin sensitivity and maintains glucose homeostasis. Recent experimental
studies strongly suggest that decreased hepatic PPAR
α
expression positively correlates
with insulin resistance and NASH severity, while NASH resolution is associated with an
upregulation of PPAR
α
as well as its target genes [
92
]. However, the evidence for the use
of selective PPARαagonists in NAFLD is limited.
Fenofibrate (200 mg/d) induced complete resolution of biochemical and ultrasono-
graphic evidence of NAFLD in almost half of the patients in an open-label RCT [
93
], but
had minimal efficacy regarding liver histology [
94
]. Gemfibrozil (600 mg/d for 4 weeks)
reduced liver enzyme levels but also did not produce any meaningful changes in liver
morphology [
95
], while clofibrate (2 g/d) failed to show any beneficial effect in NASH
patients [
96
]. Pemafibrate (0.2 mg/d) improved biochemical markers of liver damage and
steatohepatitis according to non-invasive measures in NAFLD subjects [
97
], and bezafibrate
reduced liver steatosis in obese mice with metabolic syndrome [98].
PPAR
γ
are critical positive regulators of adipocyte differentiation and lipogenesis,
insulin sensitivity, and glucose uptake by skeletal muscle. Additionally, PPAR
γ
may
reduce inflammation via inhibition of macrophage activation and tumour necrosis factor
α
production [
92
]. Latest EASL-EASD-EASO [
1
] and AASLD [
99
] clinical practice guidelines
support the use of pioglitazone, a selective PPAR
γ
agonist, in progressive and/or high-
risk, biopsy-proven NASH, due to its efficacy regarding liver histology in NASH patients
with or without T2DM. More recently, lobeglitazone has been demonstrated to reduce
Biomedicines 2022,10, 274 10 of 34
intrahepatic fat content and biochemical markers of liver damage in T2DM patients with
NAFLD [
100
]. An experimental study found nifedipine, an L-type calcium channel blocker,
to exert protective action against diet-induced NASH in rats, most probably due PPAR
γ
activation [101].
PPAR
δ
is the less studied PPAR isotype despite its ubiquitous expression. Experimen-
tal studies have reported PPAR
δ
to reduce very low-density lipoprotein cholesterol levels,
inhibit adipocyte growth and lipid uptake, prevent the formation of reactive oxygen species,
and modulate Kupffer cell activation [
92
]. A recent study found a correlation between
severe, but not moderate, hepatic steatosis and decreased hepatic PPAR
δ
expression [
102
].
Seladelpar (MBX-8025) is the only selective PPAR
δ
agonist currently in the pipeline
for the treatment of NAFLD. The interim analysis results of a 52-week phase 2b RCT of
seladelpar (10–50 mg/d) found it to reduce ALT, GGT, and ALP levels, but only minimally
influence liver steatosis at 12 weeks of treatment [
103
]. Further trials have so far focused
more on the anticholestatic properties of seladelpar, of potential value in primary biliary
cholangitis (NCT03301506).
Saroglitazar (Lipaglyn
®
), a dual PPAR
α
/
γ
agonist, is approved in India for use in
T2DM and precirrhotic NASH. In several RCT in NAFLD patients with and without T2DM,
saroglitazar (4 mg/d) was shown to significantly improve liver biochemistry as well as
hepatic steatosis by non-invasive measures over 16–24 weeks of treatment [
104
,
105
]. The
group of experimental triple PPAR
α
/
γ
/
δ
agonists is now represented by lanifibranor
(IVA337) alone, after its predecessor elafibranor (GFT-505) was discontinued due to lack
of efficacy in NAFLD in the phase 3 RESOLVE-IT trial (NCT02704403). Lanifibranor was
well tolerated and reduced liver enzyme levels and markers of fibrosis in patients with
precirrhotic, highly active NASH in the recently completed phase 2b study. The percentage
of patients with meaningful improvements in steatosis, activity, and fibrosis scores was
significantly higher in the lanifibranor-treated arms, indicating possible improvements
in hepatitis activity as well as HCB [
106
]. Two more trials to evaluate the efficacy of
lanifibranor in concomitant NAFLD and T2DM and in advanced fibrosis due to NASH are
ongoing (NCT03459079, NCT04849728).
6.2. MPC Inhibitors
In addition to their primary mechanism of action, pioglitazone and other thiazolidine-
diones are known to interact with the mitochondrial pyruvate carrier (MPC) to suppress
pyruvate transport into the mitochondrial matrix. Since this direct, non-genomic effect is
considered essential for the inhibition of hepatic gluconeogenesis by PPAR
γ
ligands, novel
MPC-inhibiting thiazolidinedione derivatives with minimal affinity towards PPAR
γ
have
been synthesized.
Azemiglitazone (MSDK-0602K) reduced liver enzyme levels in NASH/fibrosis pa-
tients regardless of T2DM presence [
107
], and was characterized by a markedly improved
safety profile [
108
]. However, the drug did not demonstrate significant effects on any of
the histological endpoints [
107
]. A phase 3 trial to evaluate azemiglitazone in diabetic or
prediabetic patients with NAFLD/NASH has been initiated (NCT03970031). An alternative
approach to minimizing the side effects of PPAR
γ
agonists is represented by PXL065, the
deuterium-stabilized R-isomer of pioglitazone [
109
], which is currently being assessed in a
phase 2 RCT (NCT04321343).
6.3. Incretin Mimetics
Incretins are a small family of intestinal L-cell-derived peptide hormones that includes
glucagon-like peptide 1 (GLP1), glucose-dependent insulinotropic polypeptide (GIP), and
oxyntomodulin. The incretin axis mediates the physiological response to hyperglycaemia
and couples glucose intake with pancreatic secretion [
110
]. GLP1 receptors (GLP1R) are
expressed in the
β
-cells, hepatocytes, white adipose tissue, brain, and skeletal muscle [
111
].
GLP1R activation induces insulin secretion, decreases insulin resistance, inhibits glucagon
release and lipogenesis, and suppresses appetite and gastrointestinal motility. Additionally,
Biomedicines 2022,10, 274 11 of 34
hepatic GLP1R stimulate FA
β
-oxidation, inhibit profibrogenic signaling pathways, and
exert a mild anti-inflammatory effect by indirectly reducing CRP, proinflammatory cytokine,
and chemokine production [112].
At the moment, semaglutide is the only GLP1R agonist being developed for the
treatment of NASH in nondiabetic subjects. In the recently completed 72-week phase 2 trial,
semaglutide treatment (0.4 mg/d) led to NASH resolution with no worsening of fibrosis
in 59% vs. 17% in the placebo group, which is considered the highest response rate that
a drug has ever achieved in a NASH trial up to now [
113
,
114
]. Currently, semaglutide is
being evaluated in several phase 2 trials as monotherapy (NCT03884075, NCT04216589)
as well as in combinations with empagliflozin (NCT04639414), and cilofexor/firsocostat
(NCT04971785). A 5-year-long phase 3 study designed to include 1200 patients with
precirrhotic NASH has been initiated (NCT04822181).
Exenatide, lixisenatide, liraglutide, and dulaglutide have all demonstrated signifi-
cant antisteatotic activity, and (with the exception of lixisenatide) improved intrahepatic
cholestasis in T2DM/NAFLD patients. Amelioration of cytolysis markers was reported
for exenatide [
115
,
116
] and dulaglutide [
117
,
118
], and lixisenatide was effective against
liver fibrosis and inflammation [
119
]. Liraglutide reduced hepatitis activity and liver fi-
brosis as well as attenuated HCB in NASH patients regardless of the presence of T2DM,
as determined by the phase 2 LEAN study [
120
]. A recent meta-analysis by Ghosal et al.,
including 8 RCT and over 600 patients, found that GLP1R agonists in general improve liver
function and histology by improving glycaemia, reducing body weight and hepatic fat
content, which in turn might be beneficial for hepatic inflammation in NAFLD concomitant
with T2DM [121].
While both GIP and GLP1 are potent insulin secretagogues, GIP has a more robust,
dose-dependent secretory pattern, and appears to make a greater contribution than GLP1 to
prandial insulin secretion in healthy subjects, while in T2M its activity is depleted. In addi-
tion, GIP, but not GLP1, stimulates glucagon secretion by the
α
-cells at low glycaemia under
physiological conditions, and in a glucose-independent fashion in T2M [
110
]. GIP receptors
(GIPR) upregulate lipogenesis, FA esterification and TAG accumulation in adipocytes, and
inhibits prandial lipid absorption. Supraphysiological levels of GIP have been associated
with increased systemic inflammatory response, and are considered a risk factor for the
development of NASH [122].
Tirzepatide (LY3298176) is a synthetic injectable dual GLP1/GIP peptide agonist
currently researched for NAFLD treatment. In T2DM subjects with NASH, tirzepatide
(
1–15 mg/week
for 26 weeks) effectively reduced ALT, AST, cytokeratin 18, Pro-C3, and
increased adiponectin levels [
123
]. Additionally, tirzepatide treatment led to greater im-
provements in liver fat content compared to titrated insulin degludec in T2DM, according
to the phase 3 SURPASS-3 RCT results [
124
]. A phase 2 study, designated SYNERGY-NASH,
has been initiated to evaluate the efficacy of tirzepatide in nondiabetic subjects with NASH
(NCT04166773).
Oxyntomodulin shares sequence similarity with both and GLP1 and glucagon, and
activates GLP1R and glucagon receptors (GCGR) under physiological conditions. Simulta-
neous GLP1R activation prevents hyperglycaemic response characteristic of glucagon, at
the same time potentiating its catabolic effects and greatly intensifying hepatic glycolysis,
glycogenolysis, and lipolysis [
125
]. Weight reduction, anorexigenic and hypoglycaemic
effects have been linked to GLP1 activation, while GCGR activation is thought to contribute
primarily to hepatic steatosis attenuation and improved mitochondrial respiration. The
clinical utility of oxyntomodulin itself is limited by a short circulatory half-life due to rapid
renal clearance and degradation by dipeptidyl peptidase 4 (DPP4) [126].
In contrast to the native hormone, synthetic oxyntomodulin mimetics are resistant to
proteolytic cleavage and have prolonged pharmacological action. Cotadutide (MEDI0382)
(100–300
µ
g/d) caused substantial improvements in liver enzyme levels and markers
of liver fibrosis in concomitant obesity, T2DM, and NASH in a phase 2b study that in-
cluded over 800 subjects [
127
]. Efinopegdutide (HM12525A, MK-6024), a PEGylated
Biomedicines 2022,10, 274 12 of 34
long-acting peptide agent, has demonstrated promising antihyperlipidaemic, antisteatotic,
and anti-inflammatory activity in mice and hamsters [
128
], and is going to be evaluated in
a phase 2 RCT in NASH with semaglutide as an active comparator (NCT04944992). Other
dual GLP1/GCCR agonists intended for use in NAFLD include pemvidutide (ALT-801)
(NCT05006885), danuglipron (PF-06882961) (in combination with ervogastat) [
129
], BI
456906 (NCT04771273), and HM14320 (a glucagon-containing combination) [
130
]. Finally,
a novel triple GLP1R/GCGR/GIPR agonist, HM15211, induced significant reductions in
liver steatosis, fibrosis, and inflammation in mice [
131
]; a phase 2 clinical trial is ongoing
(NCT04505436).
GLP2, usually not considered an incretin, is prevalent in the gastrointestinal tract,
where it promotes lipid absorption, regulates intestinal motility, mucosal morphology,
function and integrity of the intestine [
132
]. Teduglutide, a selective GLP2R agonist,
reduced liver steatosis and disease activity scores in rats, possibly by restoring normal
intestinal permeability [133].
DPP4 inhibitors represent a group of indirect incretin mimetics as they prevent the pro-
teolytic cleavage of GLP1, GIP, and oxyntomodulin. To the best of our knowledge, no DPP4
inhibitors are yet in the global pipeline for liver disease. However, a number of small-scale
clinical trials have evaluated their potential efficacy in NAFLD in the presence or absence
of concomitant T2DM. Among this group, only sitagliptin (100 mg/d) was found effective
against hepatic steatosis and HCB irrespective of T2DM in a 1-year open-label RCT [
134
].
Vildagliptin (100 mg/d) [
135
], saxagliptin (5 mg/d) [
136
], omarigliptin (25 mg/week) [
137
],
and teneligliptin (20 mg/d) [
138
] improved liver function and some non-invasive markers
of NAFLD, and alogliptin (25 mg/d) was only moderately effective against NASH over
12 months of treatment in T2DM/NAFLD patients [
139
]. A recent meta-analysis by dos
Santos et al. found the existing evidence for DPP4 inhibitors in NAFLD to be of poor
quality and altogether not supportive of their clinical effectiveness [140]. Evogliptin [141],
anagliptin [
142
,
143
], trelagliptin [
144
], gemigliptin [
145
], and linagliptin [
146
] have demon-
strated beneficial effects in experimental rodent models, but their clinical value remains to
be explored in future trials.
6.4. SGLT Inhibitors
Sodium/glucose cotransporter (SGLT) 2 inhibitors are a relatively novel class of oral
antidiabetic agents that increase urinary glucose excretion by inhibiting glucose reabsorp-
tion by SGLT2 in the proximal tubules. Several trials have demonstrated the improvement
of cardiovascular and renal outcomes by treatment with compounds of this class, namely,
empagliflozin, canagliflozin, and dapagliflozin [
147
]. SGLT2 inhibitors are known for their
multiple metabolic effects that are notably relevant to NAFLD pathophysiology, including
the general shift towards increased ketogenesis, gluconeogenesis, glycogenolysis, and FA
β
-oxidation. They inhibit leptin production by adipocytes, leading to decreased food intake,
increase adiponectin levels, provide mild insulin sensitization, suppress HSC activation
and fibrogenesis. Additionally, SGLT2 inhibitors may indirectly suppress sympathetic
innervation and increase the vagal tone, thereby preventing the activation of Kupffer cells
and the associated inflammatory processes [148,149].
Recent evidence mostly supports the efficacy of the majority of SGLT2 inhibitors for
improving liver dysfunction, steatosis and fibrosis in NAFLD concomitant with T2DM.
Among this group, only dapagliflozin, empagliflozin, and canagliflozin treatment was
associated with beneficial effects in nondiabetic NAFLD patients. Dapagliflozin (10 mg)
significantly reduced ALT, AST, and GGT levels, according to a retrospective study [
150
],
while empagliflozin (10 mg/d) also attenuated liver steatosis and liver stiffness, indicative
of potential antifibrotic activity, in a small-scale RCT [
151
]. The phase 3 DEAN trial to eval-
uate dapagliflozin in biopsy-confirmed NASH patients has been initiated (NCT03723252).
Canagliflozin (100 mg/d) improved liver enzyme levels and FIB-4 index values in an
open-label, uncontrolled pilot study [
152
]. Additionally, empagliflozin had a beneficial
effect on cognitive functions and reduced anxiety in an experimental NAFLD model [
153
].
Biomedicines 2022,10, 274 13 of 34
Ipragliflozin (50 mg/d for 72 weeks) ameliorated liver fibrosis and enhanced NASH
resolution [
154
], remogliflozin etabonate (50–1000 mg/d for 12 weeks) reduced FIB-4 and
NAFLD-fibrosis scores [
155
], and ertugliflozin (5 or 15 mg/d for 52 weeks) reduced liver
transaminase levels [
156
] in TD2M patients with different stages of NASH. Several pilot
studies in T2DM/NAFLD subjects confirmed the antisteatotic properties of luseogliflozin
and tofogliflozin [157–159].
The SGLT1 subtype plays a relatively smaller (10–20% and up to 40% when SGLT2
are blocked) role in the renal glucose reabsorption, but is more abundant in the small
intestine along with the heart and lungs. Intestinal SGLT1 (iSGLT1) inhibition leads to
substantially reduced glucose and galactose absorption from the intestinal lumen, and
increased incretin (GLP1, peptide YY) release by enteroendocrine cells [
160
]. Currently,
licogliflozin (LIK066) is the only SGLT1/2 inhibitor being evaluated in NASH independent
of T2DM presence, alone and in combination with the FXR agonist tropifexor, in the ongoing
phase 2 ELIVATE study (NCT04065841). Previously, licogliflozin (150 mg/d) reduced
ALT, ALT, GGT levels and liver fat content over 12 weeks compared to placebo [
161
]. A
novel compound, SGL5213, has been identified as a selective iSGLT1 inhibitor, and has
demonstrated insulin-sensitizing, anti-inflammatory, and antifibrotic activity in a murine
model of NAFLD [162].
6.5. α-Glucosidase Inhibitors
α
-Glucosidase is a carbohydrate hydrolase located in the brush border of the small
intestine that catalyzes the breakdown of dietary starch and disaccharides to yield glucose.
α
-Glucosidase inhibitors slow down carbohydrate digestion and absorption, thereby reduc-
ing postprandial hyperglycaemia. However, they are characterized by only modest overall
antidiabetic activity, and are not too often used in clinical practice [
163
]. Despite some
scientific interest concerning the use of this class of drugs for the treatment of liver diseases,
data regarding their efficacy for NAFLD remain scarce. Acarbose (100 mg/d) improved
AST, ALT levels and lipid profiles, albeit to a lesser extent than ezetimibe, in a 10-week
small-scale RCT in non-diabetic NASH patients [
164
]. Miglitol treatment (
150 mg/d
for
12 months) was associated with significant improvements in steatosis, lobular and portal
inflammation, and NAS scores, while fibrosis and hepatocyte ballooning remained un-
changed [
165
]. Finally, voglibose prevented hepatic steatosis in obese rats, but was slightly
inferior to empagliflozin [166].
An overview of the current drug development pipeline for NAFLD is given in Figure 1.
Biomedicines 2022,10, 274 14 of 34
Biomedicines 2022, 10, x FOR PEER REVIEW 14 of 35
Figure 1. An overview of the current drug development pipeline for non-alcoholic fatty liver dis-
ease. FASN, fatty acid synthase; DGAT, diglyceride acyltransferase; FXR, farnesoid X receptor; FGF,
fibroblast growth factor; TLR4, toll-like receptor 4; LOXL2, lysyl oxidase-like protein 2; ATX, auto-
taxin; SGLT, sodium/glucose contransporter; MPC, mitochondrial pyruvate carrier; PPAR, peroxi-
some proliferator-activated receptor; THRβ, thyroid hormone receptor β; PUFA, polyunsaturated
fatty acid; ACC, acetyl-CoA carboxylase; BUDCA, berberine ursodeoxycholate; LMS, leucine-met-
formin-sildenafil; LMSC, liver-derived mesenchymal stromal cells.
7. Other Agents
7.1. Probiotics
According to recent evidence, NAFLD pathogenesis is tightly associated with intes-
tinal bacterial overgrowth and an upset balance between Bacteroidetes and Firmicutes
species, leading to alterations in the gut-derived metabolite and endotoxin production
[167]. A number of small-scale RCT in paediatric and adult NAFLD patients have demon-
strated the beneficial effects of probiotics containing various combinations of Bifidobacte-
rium spp. [168], Lactobacillus spp. [169,170], Streptococcus thermophilus [171], Pediococcus
pentosaceus [172], Lactococcus spp., Propionibacterium spp., and Acetobacter spp. [173] Two
meta-analyses of 134 and 535 NAFLD patients from 4 and 9 RCTs, respectively, have con-
firmed that probiotics can improve insulin sensitivity, ameliorate dyslipidaemia and
Figure 1.
An overview of the current drug development pipeline for non-alcoholic fatty liver disease.
FASN, fatty acid synthase; DGAT, diglyceride acyltransferase; FXR, farnesoid X receptor; FGF,
fibroblast growth factor; TLR4, toll-like receptor 4; LOXL2, lysyl oxidase-like protein 2; ATX, autotaxin;
SGLT, sodium/glucose contransporter; MPC, mitochondrial pyruvate carrier; PPAR, peroxisome
proliferator-activated receptor; THR
β
, thyroid hormone receptor
β
; PUFA, polyunsaturated fatty
acid; ACC, acetyl-CoA carboxylase; BUDCA, berberine ursodeoxycholate; LMS, leucine-metformin-
sildenafil; LMSC, liver-derived mesenchymal stromal cells.
7. Other Agents
7.1. Probiotics
According to recent evidence, NAFLD pathogenesis is tightly associated with intestinal
bacterial overgrowth and an upset balance between Bacteroidetes and Firmicutes species,
leading to alterations in the gut-derived metabolite and endotoxin production [
167
]. A
number of small-scale RCT in paediatric and adult NAFLD patients have demonstrated the
beneficial effects of probiotics containing various combinations of Bifidobacterium spp. [
168
],
Lactobacillus spp. [
169
,
170
], Streptococcus thermophilus [
171
], Pediococcus pentosaceus [
172
],
Lactococcus spp., Propionibacterium spp., and Acetobacter spp. [
173
] Two meta-analyses of 134
and 535 NAFLD patients from 4 and 9 RCTs, respectively, have confirmed that probiotics can
improve insulin sensitivity, ameliorate dyslipidaemia and systemic inflammation, reduce in-
Biomedicines 2022,10, 274 15 of 34
trahepatic fat content, and rescue impaired liver function, overall improving the clinical out-
comes in NAFLD [
174
,
175
]. Additionally, probiotics based on Saccharomyces boulardii [
176
]
and Clostridium butyricum [
177
] have demonstrated antisteatotic, anti-inflammatory, and
antifibrotic properties in animal models of NAFLD. Clinical evidence for probiotic use in
NAFLD is summarized in Table 1.
Table 1.
Probiotic formulations with evidence of hepatoprotective activity in non-alcoholic fatty liver disease.
Composition
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Bifidobacterium longum + + ± ± [168]
Lactobacillus acidophilus + [178]
L. acidophilus,B. lactis + [170]
L. rhamnosus + [169]
L. plantarum * + [179]
L. paracasei * + + [180]
L. johnsonii * + + [181]
L. reuteri + [182]
L. delbrueckii subsp.
bulgaricus,Streptococcus
thermophilus
+ [183]
L. acidophilus,L. rhamnosus,
B. bifidum,B. lactis + + [184]
L. acidophilus,L. rhamnosus,
L. plantarum,L. delbrueckii
subsp. bulgaricus,B. bifidum
+ + [185]
L. acidophilus,L. rhamnosus,
L. paracasei,B. lactis,B. breve,
Pediococcus pentosaceus
+ [172]
L. acidophilus,L. plantarum,
L. paracasei,L. delbrueckii
subsp. bulgaricus,B. breve,
B. longum, B. infantis
±[171]
Lactobacillus spp.,
Bifidobacterium spp.,
Lactococcus spp.,
Propionibacterium spp.,
Acetobacter spp.
+ + ±+ [173]
L. acidophilus,L. plantarum,
L. delbrueckii subsp.
bulgaricus,L. casei,B. breve,
B. longum,B. infantis,
S. thermophilus
+ [186]
Clostridium butyricum * + + [177]
Saccharomyces boulardii * + + [176]
* Preclinical data given. +, definite positive effect; ±, possible positive effect; HCB, hepatocellular ballooning.
Biomedicines 2022,10, 274 16 of 34
7.2. Mesenchymal Stromal Cells
Lately, cell-based therapy has emerged as a feasible alternative for the treatment of
different stages of NAFLD. In particular, experimental evidence supports the use of bone
marrow- [
187
], umbilical cord- [
188
], and compact bone-derived mesenchymal stromal
cells [
189
] as well as hepatocytes derived by differentiating induced pluripotent stem
cells [
190
]. The crosstalk between hepatic stem cells and their possible therapeutic applica-
tion for NAFLD are discussed in detail in a recent review by Overi et al. [191].
HepaStem
®
is a first-in-class allogeneic stem cell therapy product containing human
adult liver-derived progenitor cells with potential indications including cirrhotic and
precirrhotic NASH as well as acute-on-chronic liver failure (ACLF). HepaStem
®
cells,
obtained from healthy donors, are expected to modulate the inflammatory response and
inhibit HSC activation, thereby reducing liver fibrosis. A small-scale phase 2a RCT found
HepaStem to be safe and well tolerated, and indicated potential efficacy for ACLF and/or
decompensated liver cirrhosis [192].
7.3. Fraudulent Fatty Acids
Fraudulent, or abnormal fatty acids, represented by bempedoic acid (ETC-1002) and
gemcabene (CI-1027), are molecules with structures similar to those of oleic or linolenic
acid that regulate metabolic pathways in the liver, resulting in enhanced FA catabolism.
After conversion into its active form, bempedoic acid acts as false substrate and inhibits
hepatic adenosine triphosphate citrate (pro-S)-lyase, an enzyme upstream of 3-hydroxy-
3-methylglutaryl-CoA reductase in the cholesterol synthesis pathway. This links fraud-
ulent fatty acids to statins, whose possible beneficial effects for NAFLD are reviewed
elsewhere [193].
Bempedoic acid is approved in the USA and EU as monotherapy and as a fixed-
dose combination with ezetimibe for the treatment of hypercholesterolaemia [
194
]. In a
high-fat diet-induced murine model of NASH, it caused significant reductions in ALT and
AST levels, hepatic TAG accumulation, proinflammatory and profibrotic gene expression,
resulting in improved NAFLD activity and liver fibrosis by histological analysis [195].
Gemcabene (PD-72953), a structurally optimized derivative of bempedoic acid, forms
a CoA conjugate that inhibits ACC, and reduces apolipoprotein C-III expression [
194
].
In a mouse model of NASH/HCC, it diminished micro- and macrovesicular liver steato-
sis, HCB, inflammatory infiltration, and fibrosis, which corresponded to downregulated
proinflammatory, lipogenesis, and profibrogenic marker expression [
194
]. Gemcabene
was being developed for the treatment of paediatric NAFLD, but was discontinued and
repurposed for another indication after a lack of efficacy was demonstrated in a phase 2a
proof-of-concept study (NCT03436420).
7.4. Tesamorelin
Tesamorelin (TH9507) is a growth hormone (GH) releasing hormone analogue that
is thought to stimulate lipolysis via increasing endogenous GH levels while maintaining
feedback inhibition and limiting toxicity compared to native GH. Tesamorelin reduced liver
fat content and visceral fat in a preliminary study in antiretroviral-treated patients with
human immunodeficiency virus (HIV)-associated lipodystrophy [
196
]. A phase 2 trial to
evaluate the effects of tesamorelin on liver steatosis and cardiovascular risk in obese NASH
patients is recruiting (NCT03375788), and a phase 3 study in the general population with
NAFLD including a HIV cohort has been planned [197].
7.5. Berberine Ursodeoxycholate
Berberine ursodeoxycholate (BUDCA, HTD1801) is an ionic salt of the isoquino-
line alkaloid berberine and ursodeoxycholic acid (UDCA). According to a meta-analysis
by Wei et al., berberine can significantly improve liver function, lipid profiles, and gly-
caemic control in patients with NAFLD [198] due to adenosine monophosphate-activated
protein kinase (AMPK) activation, stimulation of glycolysis, and, possibly, inhibition of
Biomedicines 2022,10, 274 17 of 34
α
-glucosidase [
199
]. UDCA, in turn, is a bile acid long used for the treatment of NASH
and chronic cholestatic diseases, whose hepatoprotective effects are confirmed by several
systematic reviews and meta-analyses [
200
,
201
]. In a phase 2 proof-of-concept RCT in
T2DM patients with presumed NASH, BUDCA reduced liver enzyme levels and liver
steatosis by MRI-PDFF [202].
7.6. Miricorilant
Miricorilant (CORT 118335) is an investigational glucocorticoid receptor agonist/antagonist
and a mineralocorticoid receptor antagonist currently in development for NASH and antipsychotic-
induced weight gain. Results of a phase 2a study in NASH patients demonstrated that mirico-
rilant (600 mg/d) effectively ameliorated liver steatosis by radiological measures. However,
miricorilant treatment was associated by transient yet significant increases in serum
transaminases [
203
], and the trial was subsequently put on hold due to safety concerns
(NCT03823703).
7.7. Nitazoxanide
Nitazoxanide (Alinia
®
) is an FDA-approved broad-spectrum thiazolide antiprotozoal
and antiparasitic agent, lately reported to be a potent AMPK activator and inhibitor of HSC
activation. In experimental studies in mice, nitazoxanide (100 mg/kg/d) attenuated dyslip-
idaemia, liver steatosis [
204
], fibrosis, inflammation, and HCB, demonstrating synergistic
effects with the pan-PPAR agonist elafibranor [
205
]. Moreover, the anti-anaerobic activity of
nitazoxanide may determine its use in preventing the recurrence of hepatic encephalopathy
as a viable alternative to rifaximin (NCT04161053) [206].
7.8. Pirfenidone
Pirfenidone (Esbriet
®
) is a pyridine derivative with antifibrotic, anti-inflammatory,
and antioxidant properties, the precise mechanisms of which are still unclear. In the liver,
pirfenidone may decrease fibronectin, TGF
β
, collagen production and attenuate fibroge-
nesis, hepatocyte necrosis, and necroinflammation [
207
,
208
]. In the phase 2 PROMETEO
study, pirfenidone (1200 mg/d) markedly reduced transaminase levels and advanced liver
fibrosis of predominantly nonalcoholic aetiology [209].
7.9. Miscellaneous
Other investigational drugs with potential therapeutic value in NAFLD include an-
tileukotriene agents [
210
], GPCR modulators [
211
], anti-IL mABs [
212
], IL22 axis mod-
ulators [
213
], purinergic receptor agonists [
214
], antioxidants [
215
], antisense oligonu-
cleotides [
216
], multitarget epigenetic regulators [
217
], and many more. A comprehensive
list of drug candidates and experimental agents with evidence of hepatoprotective activity
in NAFLD is given in Table 2.
Biomedicines 2022,10, 274 18 of 34
Table 2. Drug candidates and experimental agents with evidence of hepatoprotective activity in non-alcoholic fatty liver disease.
Name Mechanism of Action Development
Phase
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Resmetirom THRβagonist 3 + + ± ± + + [11]
VK2809 THRβagonist 2 + [12]
ASC41 * THRβagonist 2 + + + + [13]
TERN-501 * THRβagonist 1 + + + [15]
Firsocostat ** ACC inhibitor 2 + + + + + ±[23]
Clesacostat ** ACC inhibitor 2 + [24,31]
ASC40 FASN inhibitor 2 + + + + [16]
Aramchol SCD1 inhibitor 3 + + + + + [30]
Ervogastat DGAT2 inhibitor 2 + [31]
ION224 DGAT2 inhibitor 1 + ±[216]
Docosahexaenoic acid ω-3 PUFA - + [40]
Epeleuton ω-3 PUFA 2 ±[44]
Icosabutate ω-3 PUFA 2 + + ±+ [46]
Eicosapentanoic acid * ω-3 PUFA - + + + [218]
Obeticholic acid FXR agonist 3 + + + + + + [49,50]
EDP-305 FXR agonist 2 + + + + [54,56]
Tropifexor FXR agonist 2 + + + + [63]
Cilofexor FXR agonist 2 + + + [64]
Vonafexor FXR agonist 2 + + + [65]
MET409 FXR agonist 2 + + [59]
TERN-101 FXR agonist 2 + + [67]
ASC42 * FXR agonist 2 + + + [16]
INT-767 * FXR agonist 2 + + + + [58]
EDP-297 * FXR agonist 1 + + + ±[219]
BAR502 * FXR agonist - + + + [57]
GW4064 * FXR agonist - + + [220]
Biomedicines 2022,10, 274 19 of 34
Table 2. Cont.
Name Mechanism of Action Development
Phase
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Aldafermin FGF19 analogue 2 + + + [70]
Efruxifermin FGF21 analogue 2 + + ±+ + [71]
BIO89-100 FGF21 analogue 2 + + [72]
BFKB8488A FGFR1c/KLB agonist 2 + [74]
MK-3655 FGFR1c/KLB agonist 2 + [221]
GLP-1-Fc-FGF21 D1 *
FGF21 analogue, GLP1R agonist
- + + [75]
GB1211 * galectin-3 antagonist 2 + [88]
GM-CT-01 * galectin-3/1 antagonist - + + + + [79]
JKB-122 TLR4 antagonist 2 + + [83]
Eritoran * TLR4 antagonist - + + + [81]
PXS-5153A * LOXL2/3 inhibitor - + + [87]
Bezafibrate * PPARαagonist - + + ±[98]
Pemafibrate PPARαagonist - + ±+ [97]
Fenofibrate PPARαagonist - + ±+ [93,94]
Gemfibrozil PPARαagonist + [95]
Nifedipine * PPARγagonist - + + [101]
Seladelpar PPARδagonist 2 + + [103]
Saroglitazar PPARα/γagonist 2 + + + + [104,105]
Lanifibranor PPARα/γ/δagonist 3 + + + + + [106]
Pioglitazone PPARγagonist, MPC inhibitor - + + + + [222]
Lobeglitazone PPARγagonist, MPC inhibitor - + + [100]
Azemiglitazone MPC inhibitor 3 + ± ± ± [107]
PXL065 * MPC inhibitor 2 + + + [109]
Biomedicines 2022,10, 274 20 of 34
Table 2. Cont.
Name Mechanism of Action Development
Phase
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Semaglutide GLP1R agonist 3 + + [113]
Exenatide GLP1R agonist - + + + [115,116]
Lixisenatide GLP1R agonist - + + + [119]
Liraglutide GLP1R agonist - + + + + + [120]
Dulaglutide GLP1R agonist - + + + [117,118]
Teduglutide * GLP2R agonist - + ± ± [113]
Tirzepatide GLP1R/GIPR agonist 2 + + [123,124]
Cotadutide GLP1R/GCGR agonist 2 + + ±[127]
Efinopegdutide * GLP1R/GCGR agonist 2 + ± ± [128]
Pemvidutide GLP1R/GCGR agonist 1 [223]
HM15211 * GLP1R/GCGR/GIPR agonist 2 + + [131]
Sitagliptin DPP4 inhibitor - + + ±[134]
Vildagliptin DPP4 inhibitor - + + + [135]
Saxagliptin DPP4 inhibitor - + + ±+ [136]
Alogliptin DPP4 inhibitor - + [139]
Omarigliptin DPP4 inhibitor - + + + + + [137]
Teneligliptin DPP4 inhibitor - + + [138]
Evogliptin * DPP4 inhibitor - + + + [141]
Anagliptin * DPP4 inhibitor - + + + [142,143]
Trelagliptin * DPP4 inhibitor - + + + ±[144]
Gemigliptin * DPP4 inhibitor - + + + [145]
Linagliptin * DPP4 inhibitor - + + [146]
Dapagliflozin SGLT2 inhibitor - + + ±+ [150,224]
Empagliflozin SGLT2 inhibitor - + + + + + [151,225]
Canagliflozin SGLT2 inhibitor - + + + + [152]
Biomedicines 2022,10, 274 21 of 34
Table 2. Cont.
Name Mechanism of Action Development
Phase
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Ipragliflozin SGLT2 inhibitor - ±+ + [154,226]
Ertugliflozin SGLT2 inhibitor - + [156]
Remogliflozin SGLT2 inhibitor - + + [155]
Luseogliflozin SGLT2 inhibitor - + + + [157,158]
Tofogliflozin SGLT2 inhibitor - + + + + [159]
Licogliflozin ** SGLT1/2 inhibitor 2 + + + [160,161]
SGL5213 * iSGLT1 inhibitor - + + ±+ [162]
Miglitol α-glucosidase inhibitor - + + ±+ + [165]
Acarbose α-glucosidase inhibitor - + [164]
Voglibose * α-glucosidase inhibitor - + [166]
Liver-derived MSC MSC 2 + + [192]
Umbilical cord-derived MSC * MSC - + [188]
Compact bone-derived MSC * MSC - + + + + [189]
Tesamorelin GHRH analogue 3 + + [196]
Berberine ursodeoxycholate multimodal metabolic 2/1 + + + [202]
Miricorilant GR agonist/antagonist, MR
antagonist 2 + [203]
Nitazoxanide * AMPK activator 2 + + [204,205]
PXL770 AMPK activator 2 + [227]
Leucine + metformin +
sildenafil
AMPK activator, eNOS
activator 2 + [228]
Pirfenidone * multimodal antifibrotic - + + [209]
PBI-4547 * GPCR84 antagonist - + + ±[229]
CpdA * GPCR84 antagonist - + + + + [230]
CpdB * GPCR84 antagonist - + + + + [230]
GPR120 agonist III * GPCR120 agonist - + ±+ [231]
Metabolitin * GPRC6A agonist - + + + [232]
SCO-267 * GPR40 agonist - + + + [233]
Biomedicines 2022,10, 274 22 of 34
Table 2. Cont.
Name Mechanism of Action Development
Phase
Liver-Related Effects
Cytolysis Steatosis HCB Inflammation Fibrosis Cholestasis References
Evolocumab PCSK9 inhibitor - ±[234]
Alirocumab PCSK9 inhibitor - ±[234]
X203 * IL11 antagonist - + + + + [212]
X209 * IL11RA antagonist - [212]
Ezetimibe NPC1L1 inhibitor - + ±+ [235]
ORMD-0801 oral insulin 2 + [236]
GalNAc-Stk25 ASO * anti-STK25 ASO - + + + + [237]
Tipelukast * LTR antagonist, PDE3/4
inhibitor, 5-LO/LT inhibitor 2±+ + + [210]
CM-101 * CCL24 antagonist 2 + ± ± ± +±[238]
Namodenoson A3AR agonist 2 + [214,239]
PLN-1474 * αvβ1antagonist 1 + [240]
CB4211 MOTS-c analogue 1 + + [241]
CER-209 * P2Y13R agonist 1 + + [242]
DUR-928
multitarget epigenetic regulator
2 + + + + [217]
CRV431 cyclophilin A/B/D inhibitor 2 + [243]
LPCN 1144 androgen receptor agonist 2 + + + + [244]
Osteocalcin * N/A - + + + + [245]
* Preclinical data given; ** developed in combination; -, not in development. +, definite positive effect;
±
, possible positive effect. HCB, hepatocellular ballooning; THR
β
, thyroid
hormone receptor
β
; ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase 1; DGAT2, diglyceride acyltransferase 2; PUFA, polyunsaturated fatty acid;
FXR, farnesoid X receptor; FGF, fibroblast growth factor; FGFR1c/KLB, FGF receptor/
β
-klotho complex; TLR4, toll-like receptor 4; LOXL, lysyl oxidase-like protein; PPAR, peroxisome
proliferator-activated receptor; MPC, mitochondrial pyruvate carrier; GLP1R, glucagon-like peptide 1 receptor; GIPR, glucose-dependent insulinotropic polypeptide receptor; GCGR,
glucagon receptor; DPP4, dipeptidyl peptidase 4; SGLT, sodium/glucose cotransporter; iSGLT1, intestinal SGLT 1; MSC, mesenchymal stromal cells; GHRH, growth hormone releasing
hormone; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; AMPK, adenosine monophosphate-activated protein kinase; eNOS, endothelial nitric oxide synthase; GPCR,
G protein-coupled receptor; GPRC6A, G protein-coupled receptor family C group 6 member A; PCSK9, proprotein convertase subtilisin/kexin type 9; IL11, interleukin 11; IL11RA,
IL11 receptor
α
subunit; NPC1L1, Niemann-Pick C1-like protein 1; STK25, serine/threonine kinase 25; ASO, antisense nucleotide; LTR, leukotriene receptor; PDE, phosphodiesterase;
5-LO/LT, 5-lipoxygenase/leukotriene pathway; CCL24, C-C motif chemokine ligand 24; A3AR, A3 adenosine receptor; MOTS-c, mitochondrial open reading frame of the twelve S
ribosomal ribonucleic acid-c; P2Y13R, P2Y purinergic receptor 13.
Biomedicines 2022,10, 274 23 of 34
8. Future Directions
Other biomolecules and pathways most recently identified as potential therapeutic
targets in NAFLD have been highlighted in many comprehensive review articles [
246
–
249
].
Of those, the sirtuins, a family of highly conserved histone and protein deacetylases, can be
considered of special interest regarding future therapeutic concepts for NAFLD. Sirtuins act
as NAD
+
-sensing signaling proteins to facilitate stress response [
250
], maintain homeostasis
during acute and chronic inflammatory response [
251
], and partake in the regulation of
energy metabolism, redox balance, cell cycle, and suppression of tumorigenesis [252].
Sirtuin 1 (SIRT1), the SIRT1/NF-
κ
B axis, SIRT3, and SIRT4 play a major role in regulat-
ing hepatic lipid metabolism, controlling oxidative stress, and mediating chronic inflamma-
tion in NAFLD and alcoholic fatty liver disease [
253
–
255
]. Accordingly, SIRT1 activation
by the polyphenol resveratrol and several small molecules have been shown to provide
protection against NAFLD and T2DM in rodent models [
253
,
256
], and may be beneficial
in human metabolic disease [
257
]. SIRT2, SIRT3, and SIRT4 upregulation induced by an
investigational molecule also prevented the progression of hepatic steatosis and fibrosis
in obese rats [
258
]. While still at early research and development stages, selective sirtuin
activators and inhibitors are considered a promising group of drug candidate molecules
with primarily anti-inflammatory mode of action [251,254,257].
9. Conclusions
Being the most prevalent cause of chronic liver disease worldwide, NAFLD is at the
same time one of the greatest areas of unmet medical need in terms of availability and
adequacy of pharmacotherapeutic options. The modern pipeline for NAFLD includes
a plethora of drug candidates with diverse and innovative mechanisms of action, al-
though reported evidence on their clinical effectiveness has so far been limited. Upcoming
treatment approaches to different stages of NAFLD include the modulation of nuclear
receptor activity in order to maintain lipid and glucose homeostasis, stimulation of bile
acid metabolism, and direct inhibition of fibrogenesis, along with a few less explored but
highly promising options.
Despite several investigational agents being seemingly close to regulatory approval
as monotherapies, novel therapeutic strategies for NAFLD will likely involve the use
of multitarget drugs or rational drug combinations. Given the exceptionally complex
pathophysiology and the multifaceted nature of this disease, NAFLD pharmacotherapy
can be expected to remain a priority for biomedical research in the nearest future.
Author Contributions:
Data analysis, writing—original draft preparation, visualization, V.A.P.;
Conceptualization, writing—review and editing, N.N.B. and S.V.O. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by Analytics and Devices Ltd. (St. Petersburg, Russia).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Part of the data analyzed in this study are openly available at Clinical-
Trials.gov, https://www.clinicaltrials.gov/ct2/home (accessed on 10 December 2021).
Conflicts of Interest: The authors declare no conflict of interest.
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