Korean J Physiol Pharmacol 2019;23(6):459-466

https://doi.org/10.4196/kjpp.2019.23.6.459

Original Article

Efficacy of evogliptin and cenicriviroc against nonalcoholic

steatohepatitis in mice: a comparative study
Zheng Wang1, Hansu Park2, and Eun Ju Bae3,*
1
College of Pharmacy, Woosuk University, Wanju 55338, 2Dong-A Socio Research Center, Dong-A ST Co., Ltd., Yongin 17073, 3College of Pharmacy, Chonbuk
National University, Jeonju 54896, Korea

ARTICLE INFO
ABSTRACT Dipeptidyl peptidase (DPP)-4 inhibitors, or gliptins, are a class of oral
Received March 26, 2019
Revised July 15, 2019
hypoglycemic drugs that have been widely used as a second-line treatment for type
Accepted August 26, 2019 2 diabetes. Gliptins, which were introduced for clinical use a decade ago, have been
shown to be beneficial against nonalcoholic fatty liver disease/nonalcoholic steato-
*Correspondence hepatitis (NASH) in animals and humans. Cenicriviroc (CVC), a dual antagonist of C-C
Eun Ju Bae chemokine receptor type 2 and 5, is currently under investigation against NASH and
E-mail: ejbae7@jbnu.ac.kr fibrosis. It was previously discovered that evogliptin (EVO) reduces hepatic steatosis
in diet-induced obese animals but the effectiveness of EVO on NASH remains un-
Key Words explored. Here, we compared the effectiveness of EVO and CVC against NASH and
Cenicriviroc fibrosis in mice fed a high-fat and high-fructose diet (HFHF). Biochemical and histo-
Dipeptidyl peptidase-4 inhibitor
logical analyses showed that mice fed a HFHF for 20 weeks developed severe hepatic
Evogliptin
Fibrosis steatosis and inflammation with mild fibrosis. Administration of EVO (0.2% wt/wt) for
Nonalcoholic steatohepatitis the last 8 weeks of HFHF feeding significantly reduced hepatic triglyceride accumula-
tion, inflammation, and fibrosis as well as restored insulin sensitivity, as evidenced
by lowered plasma insulin levels and the improvement in insulin tolerance test
curves. Treatment of mice with CVC (0.1% wt/wt) inhibited hepatic inflammation and
fibrogenesis with similar efficacy to that of EVO, without affecting hepatic steatosis.
CVC treatment also reduced plasma insulin concentrations, despite no improvement
in insulin tolerance. In conclusion, EVO administration efficiently ameliorated the
development of NASH and fibrosis in HFHF-fed mice, corroborating its therapeutic
potential.

INTRODUCTION NASH is increasing and urgent.

Dipeptidyl peptidase (DPP)-4 inhibitors enhance the biological
Nonalcoholic fatty liver disease (NAFLD) is the most common half-life of incretins such as glucagon-like peptide 1 by inhibiting
liver disease and often progresses to nonalcoholic steatohepatitis its degrading enzyme DPP-4 and thereby increase insulin secre-
(NASH). NASH ranges from simple steatosis to inflammation tion from pancreatic beta cells. These inhibitors are currently
and fibrosis and can deteriorate to cirrhosis. NAFLD/NASH is used for the management of type 2 diabetes since they were first
usually associated with obesity-linked insulin resistance and type introduced around 10 years ago. DPP-4 inhibitors have also been
2 diabetes. Due to the rapidly growing incidence of obesity and observed to have beneficial effects for the treatment of NAFLD/
NAFLD/NASH and their huge impact on global health, the ne- NASH in animal models [1-4], although their effects in humans
cessity for the development of novel therapeutic medicine against remain controversial. For instance, sitagliptin treatment to diet-

This is an Open Access article distributed under the terms Author contributions: Z.W. and H.P. performed experiments and ana-
of the Creative Commons Attribution Non-Commercial lyzed the data. E.J.B. designed the experiments, interpreted the data, and
License, which permits unrestricted non-commercial use, distribution, and wrote the manuscript. All the authors reviewed the manuscript.
reproduction in any medium, provided the original work is properly cited.
Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827

www.kjpp.net 459 Korean J Physiol Pharmacol 2019;23(6):459-466

460 Wang Z et al

induced obese mice reduced hepatic steatosis with attenuation 1-isobutyl-N-(4-(((1-propyl-1H-imidazol-5-yl)methyl)sulfinyl)

of gene expression associated with de novo lipogenesis, but up- phenyl)-1,2,3,4-tetrahydrobenzo[b]azocine-5-carboxamide)
regulated fatty acid oxidation genes [5]. It has been shown that methanesulfonic acid salt was gratefully provided by Allergan/
administration of sitagliptin 100 mg once daily for 1 year amelio- Tobira Therapeutics. Primary antibodies of α-SMA (ab5694),
rated NAFLD activity scores (NAS) by improving steatosis and F4/80 (ab6640) and CD11b (ab13357) were purchased from Ab-
ballooning, irrespective of the glucose-lowering effects against cam (Cambridge, UK), actin (sc-1616) was purchased from Santa
diabetes [6]. Contrary to this, another study observed that sita- Cruz Biotechnology (Santa Cruz, CA, USA). D(-)-Fructose was
gliptin did not cause reduction of steatosis [7,8]. Evogliptin (EVO) purchased from Samchun Pure Chemicals (Seoul, Korea).
is a selective DPP-4 inhibitor that received its first global approval
in South Korea in 2015 for the treatment of type 2 diabetes [9,10]. Animals and treatment
A recent study has shown that EVO has a protective effect against
hepatic steatosis in diet-induced obese mice [11]. However, wheth- Six week-old male C57BL/6 mice were purchased from Sam-
er EVO is effective in the prevention or treatment of NASH and tako Bio (Osan, Korea) and acclimatized for a week. Mice were
fibrosis remains to be explored. fed a normal chow diet (NCD, 13.5% fat; LabDiet, St. Louis, MO,
Cenicriviroc (CVC) was originally developed for the treatment USA) or HFHF (a 60% fat diet with 30% fructose in water) for 12
of human immunodeficiency virus [12,13], but has now gained at- weeks and further maintained on the same diet or HFD-drug
tention due to its anti-fibrosis effect in mice, rats, and humans [14]. admixture for EVO (0.2% wt/wt, a target dose of 200 mg/kg/day)
CVC acts as a dual antagonist of C-C chemokine receptors type 2 or CVC (0.1% wt/wt, a target dose of 100 mg/kg/day) for the fur-
and 5 (CCR2/CCR5), which are associated with liver inflamma- ther 8 weeks. At the end of 20 week-study, mice were euthanized
tion and fibrosis [15,16]. CCR2/CCR5 and their respective ligands after 6 h fasting and blood and tissues were harvested for analysis.
CCL2 or monocyte chemotactic protein (MCP)-1 and CCL5 play All experimental procedures were approved by the Institutional
an important role in immune cell migration and tissue infiltra- Animal Care and Use Committee of Woosuk University (approval
tion in NASH pathology. A recent report of a randomized phase No. Woosuk 2017-007).
2 clinical study performed for two years indicated that CVC has
the ability to reduce hepatic inflammation and fibrosis in NASH Biochemical analysis
patients [17]. Therefore, to date, CVC along with obeticholic acid
which is a farnesoid X nuclear receptor ligand and elafibranor Plasma levels of alanine aminotransferase (ALT), aspartate
which is an agonist of the peroxisome proliferator-activated aminotransferase (AST), triglyceride (TG) (Asan Pharmaceutical,
receptor-α and -δ, are the only agents that have been clinically Seoul, Korea), insulin (Millipore, Billerica, MA, USA) and TNFα
shown to improve fibrosis in NASH, while many other pharma- (Invitrogen, Carlsbad, CA, USA) were measured using specific
cological treatments such as gliptins and pioglitazone are cur- kits. For liver TG quantification, tissues were homogenized and
rently undergoing evaluation [18-22]. extracted in a mixture of chloroform, methanol and distilled wa-
Based on previous findings, it was hypothesized that EVO ter (2/1/1 ratio) and liver TG contents measured using a TG assay
could prevent the development of NASH by influencing hepatic kit were expressed as mg of TG per 100 mg of liver tissue. Insulin
lipid metabolism. Experiments were subsequently performed to sensitivity was also assessed by homeostasis model assessment for
compare the effect of EVO with that of CVC in high-fat and high- insulin resistance (HOMA-IR, [26 × fasting insulin level (ng/ml)
fructose diet (HFHF)-fed mice—a well-established mild NASH × fasting glucose level (mg/dl)/405]) [23]. DPP-4 activity in plasma
model. To this end, 7-week-old C57BL/6 mice were fed a HFHF and liver tissues were measured at Dong-A Socio Research Center
for 20 weeks and fed EVO or CVC as a diet-admixture for the as described previously [24].
last 8 weeks of HFHF feeding. It was demonstrated that EVO
prevented the development of NASH in these mice by suppress- Glucose (GTT) and insulin tolerance test (ITT)
ing not only steatosis but also liver inflammation and fibrosis and
that the latter two effects were superior to those of CVC. Intraperitoneal GTT (glucose 1 g/kg of body weight) and ITT
(insulin 0.75 U/kg of body weight) were performed in mice after
16 h and 6 h of fasting, respectively. The levels of blood glucose in
METHODS tail vein blood sample were measured at 0, 15, 30, 60, and 120 min
following glucose or insulin injection.
Materials
Histology
EVO ((R)-4-[(R)-3-amino-4-(2, 4, 5-trifluorophenyl)butanoyl]-
3-(t-butoxymethyl) piperazin-2-one) L-tartrate salt was synthe- For histological examination, a part of liver tissue was fixed
sized in Dong-A ST. CVC ((S,E)-8-(4-(2-butoxyethoxy)phenyl)- with 3.7% neutrally buffered formaldehyde and embedded in

Korean J Physiol Pharmacol 2019;23(6):459-466 https://doi.org/10.4196/kjpp.2019.23.6.459

Effects of evogliptin and cenicriviroc on NASH 461

paraffin. Tissue sections (5 µm) were stained with hematoxylin hexamer primer provided in the first-strand cDNA synthesis kit
and eosin (H&E) or Sirius red using standard methods for light (Applied Biosystems, Foster City, CA, USA). Specific primers for
microscopy observation. For immunohistochemical staining, each gene were designed using qPrimerDepot (https://pga.mgh.
sections were incubated overnight at 4°C with antibodies against harvard.edu/primerbank/) and described in Table 1. qPCR was
F4/80 or CD11b (Abcam) followed by incubation with second- performed as previously described using an ABI7000 and Strata-
ary antibodies for 1 h at room temperature. Liver inflammation gene3000 MXP PCR cycler with the SybrGreen detection system
in liver biopsies was graded using a modified histologic activity 6. The mRNA expression of all genes tested is normalized to the
index [25]. Results of stained sections were quantified by Image J Rps3 gene expression.
(National Institute of Health, Bethesda, MD, USA) and expressed
as percentage of positive cells out of total area. To assess the mor- Statistical analysis
phological changes in liver, NAS was determined by the degree of
steatosis (0–3), lobular inflammation (0–3) and hepatocyte bal- The values presented are expressed as the mean ± standard
looning (0–2) according to the definition of Kleiner et al. [26]. deviation (SD). The statistical significance of the differences
between treatment groups was determined by one-way ANOVA
Western blot followed by Fisher’s post-hoc analysis using GraphPad Prism 5.2
(San Diego, CA, USA). A p-value of less than 0.05 was considered
Liver tissue lysates were prepared and western blot analysis significant.
was performed as described previously [27]. Briefly, liver tissues
were homogenized in tissue protein extraction reagent (Thermo,
Waltham, MA, USA) supplemented with inhibitors of proteinase RESULTS
and phosphatase. Protein concentrations in tissue lysates were
determined by a Bio-Rad Protein Assay (Bio-Rad Laboratories, Improvement in insulin sensitivity by administration
Hercules, CA, USA). The aliquots of lysates were separated by 6% of EVO in mice fed a HFHF
or 12% sodium dodecyl sulfate-polyacrylamide gels electropho-
resis. The separated proteins were transferred to nitrocellulose C57BL/6 mice were fed a NCD or HFHF with or without
membranes. The membranes were blocked for 30 min at room EVO or CVC for a total of 20 weeks (drug treatment for the last
temperature in Tris-buffered saline containing 1% Tween 20 and 8 weeks). Body weight and food and water intake were measured
4% skim milk and incubated with the primary antibodies against weekly. Body weight was significantly higher in mice fed HFHF
interest proteins, followed by incubation with secondary antibod- than in mice fed NCD during the entire drug treatment period,
ies. Immunoreactive protein was visualized by ECL chemilumi- but decreased in mice treated with EVO despite no change in food
nescence detection kit (Amersham Biosciences, Buckingham- intake (Table 2). Given obesity accompanies insulin resistance, we
shire, UK). Image was obtained using ChemiDoc XRS+ System performed GTT and ITT 8 weeks after drug administration. Un-
(Bio-rad, Hercules, CA, USA). expectedly, glucose tolerance was preserved in the HFHF group
(Fig. 1A). However, insulin resistance as assessed by ITT devel-
RNA isolation and real-time quantitative RT-PCR oped in the HFHF control group, but was ameliorated by EVO
(qPCR) treatment (Fig. 1B). EVO also markedly decreased plasma levels
of insulin and consequently HOMA-IR, a surrogate assessment of
Total RNA was extracted from liver tissues with TRIzol reagent insulin resistance, compared to those of the HFHF control group
(Invitrogen). First-strand cDNA was generated using the random (55.52 vs. 22.77) (Fig. 1C and Table 2). Although GTT and ITT

Table 1. RT-qPCR primer sequences

Gene Forward primer Reverse primer Accession No

Ccl2 5’-ATTGGGATCATCTTGCTGGT-3’ 5’-CCTGCTGTTCACAGTTGCC-3’ NM_011333

Il6 5’-TTGGGAGTGGTATCCTCTGTGA-3’ 5’-CCACGGCCTTCCCTACTTC-3’ NM_031168
Tnfa 5’-AGGGTCTGGGCCATAGAACT-3’ 5’-CCACCACGCTCTTCTGTCTAC-3’ NM_013693
Il1b 5’-GGTCAAAGGTTTGGAAGCAG-3’ 5’-TGTGAAATGCCACCTTTTGA-3’ NM_008361
Ccl5 5’-CCACTTCTTCTCTGGGTTGG-3’ 5’-GTGCCCACGTCAAGGAGTAT-3’ NM_013653
Col2a1 5’-TAGGCCATTGTGTATGCAGC-3’ 5’-ACATGTTCAGCTTTGTGGACC-3’ NM_007742
Tgfb1 5’-GTGTGGAGCAACATGTGGAACTCTA-3 5’-TTGGTTCAGCCACTGCCGTA-3’ NM_011577
Fasn 5’-TGATGTGGAACACAGCAAGG-3’ 5’-GGCTGTGGTGACTCTTAGTGATAA-3’ NM_007988
Rps3 5’-AATGAACCGAAGCACACCATAG-3’ 5’-ATCAGAGAGTTGACCGCAGTTG-3’ NM_012052

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 462 Wang Z et al

Table 2. Body weight, food intake, liver weight, and plasma biomarkers

HFHF
Parameter NCD
Control EVO CVC
##
Body weight (g) 36.04 ± 2.87 53.30 ± 2.80*** 48.96 ± 1.61 53.02 ± 2.68
Food intake (g/day) 3.16 ± 0.26 1.98 ± 0.39*** 1.81 ± 0.25 2.12 ± 0.36
Plasma DPP-4 activity (nmol/min/ml) 2.87 ± 0.22 6.15 ± 0.42*** 0.215 ± 0.08### 6.02 ± 0.95
Liver DPP-4 activity (nmol/min/ml) 4.65 ± 0.15 3.76 ± 0.24*** 0.683 ± 0.14### 2.90 ± 0.29
Liver weight (g) 1.40 ± 0.09 3.24 ± 0.36*** 2.42 ± 0.27## 3.04 ± 0.37
Liver to body weight ratio (%) 0.04 ± 0.00 0.06 ± 0.00*** 0.04 ± 0.02## 0.06 ± 0.00
HOMA-IR 4.14 ± 2.00 55.52 ± 24.31*** 22.77 ± 13.20### 26.10 ± 12.50###
ALT (U/l) 24.23 ± 8.96 48.36 ± 17.11*** 29.74 ± 9.65## 37.16 ± 10.42
AST (U/l) 19.40 ± 4.91 34.51 ± 12.70** 32.87 ± 12.18 42.88 ± 6.74

Seven week old C57BL/6 mice were fed NCD or HFHF for 20 weeks. Mice treated with drug were fed on high-fat diet admixture
containing EVO (0.2% wt/wt) or CVC (0.1% wt/wt) for the last 8 weeks of HFHF feeding. Results are expressed as mean ± SD (n = 9–12/
group). NCD, normal chow diet; HFHF, high-fat and high-fructose diet; EVO, evogliptin; CVC, cenicriviroc; HOMA-IR, homeostasis
model assessment for insulin resistance; AST, aspartate aminotransferase; ALT, alanine aminotransferase. **p < 0.01 and ***p < 0.001 vs.
NCD; ##p < 0.01 and ###p < 0.001 vs. HFHF control.

A C
A NCD C
HFHF
500
HFHF+EVO 50 5 ***
Blood glucose

400 HFHF+CVC
40
AUC (1000)

Insulin (ng/ml)
4
(mg/dl)

300
30 3 ##
200 20 2
##

100 10 1
0 0 0
0 30 60 90 120 NCD - EVO CVC NCD - EVO CVC
Time (min) HFHF HFHF
B B
250
*** 20
Blood glucose

200 ***
AUC (1000)

## 15 #
(mg/dl)

150
100 ## 10
##
50 5

0 0
0 30 60 90 120 NCD - EVO CVC
Time (min) HFHF

Fig. 1. Glucose tolerance test (GTT) and insulin tolerance test (ITT) in mice fed a normal chow diet (NCD) or high-fat and high-fructose diet
(HFHF) with or without evogliptin (EVO) or cenicriviroc (CVC). (A) GTT (1 g/kg glucose i.p.) and (B) ITT (0.75 U/kg insulin i.p.) were performed at 8
weeks after drug administration in mice. Area under the curve (AUC) were shown. (C) Plasma levels of insulin were analyzed by ELISA. Values are mean
± SD; n = 9–12 per group. ***p < 0.001 vs. NCD; #p < 0.05 and ##p < 0.01 vs. HFHF.

were not altered by CVC treatment, plasma insulin levels were Effect of administration of EVO or CVC on the hepatic
significantly decreased by CVC treatment compared to those in steatosis, inflammation and NAS
the HFHF control group (2.08 vs. 4.20 ng/ml) with the inhibition
of HOMA-IR. These results indicate that EVO has a consistent After 8-week treatment with EVO or CVC, mice were sacri-
effect in improving insulin sensitivity in various diabetes models ficed and tissue weight and blood parameters were analyzed.
and that CVC may have potential to reduce hyperinsulinemia in Liver weight and TG amounts were higher in HFHF control mice
subjects with elevated basal plasma insulin levels. but were decreased following EVO treatment (Table 2 and Fig.
2A), suggesting that hepatic lipid accumulation was attenuated by
EVO. Indeed, H&E staining of liver sections indicated that fat ac-

Korean J Physiol Pharmacol 2019;23(6):459-466 https://doi.org/10.4196/kjpp.2019.23.6.459

Effects of evogliptin and cenicriviroc on NASH 463

A
A
B
B NCD HFHF HFHF+EVO HFHF+CVC
5

(% of positive area)
(mg/100 mg liver tissue)
200
**

F4/80+ staining
*** 4
150 # H&E
Liver TG

3
100 # #
2
50
1
0
NCD - EVO CVC F4/80 0
NCD - EVO CVC
HFHF HFHF
10

(% of positive area)
100 **
Plasma TG (mg/dl)

80 8
F4/80

Sirius red
## ##
60 CD11b 6
40 4 ##
##
20
2
0
NCD - EVO CVC 0
Sirius red NCD - EVO CVC
HFHF
HFHF

C
C
D
D
E
E
NCD HFHF HFHF+EVO HFHF+CVC
NCD
20 HFHF α-SMA
15 HFHF+EVO
Relative mRNA level

HFHF+CVC Actin
10 **##
15 5 ** 2.5
*** ** #
* ** ***

Relative intensity
2 2.0
TNF (pg/ml)

## ###
##
10
* * 1.5
## 1 * ## ## 1.0
5 ## ##
## ##
0.5
0
0 0.0
NCD - EVO CVC NCD - EVO CVC
b

1
fa

s
Il6
2

Fa
Il1

fb
cl

cl

2a
Tn
C

Tg HFHF
ol

HFHF
C

Fig. 2. Liver histology and gene expression analysis for fibrosis and inflammatory genes. (A) Triglyceride (TG) concentrations in liver and plasma
were measured with a specific TG assay kit (n = 10). (B) Representative microphotographs of liver sections stained with hematoxylin eosin (H&E), Sirius
red or antibodies against F4/80 or CD11b. The number of F4/80-positive cells were counted and expressed as a percentage of total hepatocytes (n =
4). The Sirius red–positive area was determined with Image J (n = 4). Original magnification ×200. Scale bars = 250 μm. (C) Plasma levels of TNFα were
measured using specific ELISA kits (n = 10). (D) mRNA expression for inflammatory genes, cytokines and chemokines was determined by qPCR (n = 9–12
per group). (E) Expression level of α-smooth muscle actin (α-SMA) protein was determined by western blot in liver tissues. Representative blot and
quantification results are shown (n = 9–12 per group). NCD, normal chow diet; HFHF, high-fat and high-fructose diet; EVO, evogliptin; CVC, cenicrivi-
roc. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. NCD; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. HFHF.

cumulation in the liver was elevated by the HFHF but was signifi- and macrophage infiltration, confirming its anti-inflammatory
cantly reduced in EVO-treated mice (Fig. 2B). CVC-treated mice role in NASH. However, plasma ALT and AST levels did not dif-
did not show any difference in liver fat accumulation as assessed fer between CVC-treated and HFHF control mice.
using liver TG levels and H&E staining readout. Immunohisto- The hallmarks of nonalcoholic steatohepatitis are accumula-
chemistry revealed increased accumulation of F4/80+ and CD11b+ tion of lipid droplet, lobular inflammation and ballooning de-
cells in liver of mice fed HFHF whereas normalization with EVO generation of hepatocytes. Liver TG contents and H&E-stained
or CVC treatment (Fig. 2B). liver sections showed HFHF-fed mice demonstrated hepatic ste-
The plasma level of TNFα, a highly pro-inflammatory cyto- atosis, immune cell infiltration, and hepatocellular ballooning in
kine that plays a critical role in liver inflammation, was mea- HFHF-fed control mice, thereby elevating NAS (Figs. 2A, B, and
sured. HFHF increased TNFα levels by 7-fold but co-treatment 3). However, the NAS value was significantly lower in the EVO
with EVO reduced TNFα levels to those observed in lean NCD- group, confirming the role of EVO in the prevention of NASH
fed mice (Fig. 2C). Moreover, mRNA expression of interleukin progression. CVC also reduced NAS compared to that of the
(IL)-6 (Il6 ) and MCP-1 (Ccl2) as well as TNFα (Tnfa) was sig- HFHF control group, but to a lesser extent than EVO.
nificantly reduced in EVO-treated mice compared to that in
HFHF control mice (Fig. 2D). Liver tissues from HFHF-fed mice Improvement of NASH associated fibrosis by EVO
exhibited increased macrophage infiltration, but EVO mark- administration
edly decreased this (Fig. 2B–D) by reducing proinflammatory
cytokine and chemokine levels. Reduced hepatic inflammation Sirius Red staining was used to assess whether EVO treat-
by EVO also caused a significant decrease in plasma ALT levels ment improved liver fibrosis. Compared with NCD mice, HFHF
(Table 2), which were increased in HFHF controls, indicating control mice showed collagen deposition increase by more than
hepatocyte injury in these mice. As expected, CVC treatment also 6-fold (Fig. 2B), but EVO treatment reversed this effect. These
reduced plasma levels of TNFα, expression of IL-6 and MCP-1, findings were further supported by the increase in the levels of

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464 Wang Z et al

reduce liver steatosis, inflammation, or fibrosis in various models,

NAFLD activity score

8 this study is the first to directly compare the effect a DPP-4 in-
hibitor with that of a clinically proven anti-fibrotic agent. It is pro-
6 **
## posed that EVO exerts beneficial effects against NASH and liver
fibrosis with better efficacy to that of CVC in HFHF-fed mice.
4 NASH has a very complex pathogenesis, developing from
simple steatosis and characterized by hepatocyte death, inflam-
2 mation, and varying degree of fibrosis [29]. Multiple mechanisms
are involved in the progression from simple steatosis to NASH or
0 NASH-associated fibrosis: (i) effects of inflammatory cytokines
NCD - EVO CVC and chemokines, mainly those derived from obese and inflamed
adipose tissue; stimulation of hepatic inflammation; (ii) produc-
tion of reactive oxygen species from accumulated hepatic lipid
HFHF
promotes metabolic dysfunction, lipotoxicity; and (iii) obese gut
Fig. 3. Nonalcoholic fatty liver disease (NAFLD) activity scores (NAS) leaks lipopolysaccharides (LPS) from the gut microbiome, which
was determined by the degree of steatosis, lobular inflammation
aggravates hepatic inflammation, lipid accumulation, hepatocyte
and hepatocyte ballooning after 8 week feeding of evogliptin
(EVO) or cenicriviroc (CVC) in 20 week high-fat and high-fructose apoptosis, and HSC activation. In the current study, it was found
diet (HFHF) study. Data are mean ± SD (n = 9 per group). **p < 0.01 vs. that EVO reduced body weight gain (approximately 8% less than
NCD; ##p < 0.01 vs. HFHF. that of HFHF control mice) without change in food intake, be-
ing consistent with previous results [11]. Thus, adipose-derived
proinflammatory cytokines and chemokines such as TNFα, IL-6,
collagen 1 (Col2a1) mRNA and α-SMA protein, markers of acti- and MCP-1 were markedly reduced in the plasma or liver by EVO
vated hepatic stellate cell (HSC), in HFHF controls; these effects administration. Consequently, TNFα stimulation of liver inflam-
were significantly reduced by EVO treatment as well (Fig. 2D, E). mation may be suppressed, which is reflected by reduced NAS.
CVC significantly reduced Sirius red-positive area and α-SMA The impact of TNFα on NASH development and progression
expression to a similar degree as EVO. is very diverse, leading to apoptosis and impairment of insulin
signaling in hepatocytes and also the activation of immune cells
and HSC. In this study, complete normalization of plasma levels
DISCUSSION of TNFα by EVO treatment might prevent not only hepatic in-
flammation, insulin resistance, and hepatocyte apoptosis but also
In this report, using a HFHF-induced murine NASH model HSC activation, and thus fibrogenesis. A recent study showed that
with fibrosis and insulin resistance, it was shown that EVO not vildagliptin inhibited DPP-4 activity in gut microbiota leading to
only improves insulin sensitivity but also ameliorates NASH and improvement in hepatic immunity by suppressing LPS produc-
fibrosis with a superior efficacy to that of CVC, an anti-NASH/ tion from gut microbiota [30]. As DPP-4 activity both in the plas-
fibrosis drug under clinical evaluation. It was also confirmed that ma and the liver was completely repressed in mice treated with
EVO significantly prevented HFHF-induced hepatic steatosis, EVO (Table 2), it is possible that DPP-4 activity in gut microbiota
which is consistent with a previous study using HFD-fed mice and thus LPS production might be also suppressed in these mice,
[11]. In contrast to EVO, CVC did not show a pronounced im- which could contribute to the protection from hepatic inflamma-
provement in steatosis as observed in the HFHF control group, tion.
although it ameliorated hepatic inflammation and fibrosis. These It was also observed that hepatic accumulation of F4/80-
results are somewhat unexpected because the previous study positive macrophages was almost completely normalized by EVO
has reported that CVC treatment (20 and 100 mg/kg/day, p.o.) with less CD11b-positive infiltrates. This indicates reduced infil-
in mice with NASH improved NAS with reduction in steatosis tration of circulating monocytes into liver tissues, and is attribut-
as well as lobular inflammation and hepatocyte ballooning [28]. ed to less chemokine stimulation in combination with repressed
These differences might be derived from multiple reasons such activation of Kupffer cells, which may contribute to the preven-
as different models of NASH (streptozotocin injection plus HFD tion of liver inflammation. The infiltration of immune cells and
feeding vs. HFHF), duration of HFD feeding and drug treatment subsequent inflammation are key steps to induce or modify
(total 5 week HFD feeding with 3 week of CVC treatment vs. total fibrogenesis. For instance, deficiency of CCR2 or CCR5 has been
20 week HFHF feeding with 8 week of CVC treatment). shown to prevent NASH or fibrosis [16,31]. In this regard, CVC as
Similarly to EVO, HOMA-IR was also improved by CVC due to a CCR2/5 antagonist has been shown to have anti-fibrotic activ-
lowered plasma insulin levels, which has not been demonstrated ity in various animal models and patients [17,28]. CVC-mediated
earlier. Although DPP-4 inhibitor members have been shown to CCR2 antagonism is thought to reduce recruitment, migration,

Korean J Physiol Pharmacol 2019;23(6):459-466 https://doi.org/10.4196/kjpp.2019.23.6.459

Effects of evogliptin and cenicriviroc on NASH 465

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to additionally impair the migration, activation, and proliferation 3. Yilmaz Y, Yonal O, Deyneli O, Celikel CA, Kalayci C, Duman DG.
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ings warrant further investigation using additional doses and
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ACKNOWLEDGEMENTS 11. Kim MK, Chae YN, Ahn GJ, Shin CY, Choi SH, Yang EK, Sohn YS,
Son MH. Prevention and treatment effect of evogliptin on hepatic
This work was supported by grants from Dong-A ST Co., Ltd. steatosis in high-fat-fed animal models. Arch Pharm Res. 2017;40:
and in part by grants from the Basic Science Research Program 268-281.
(2017R1A2B4008593) through the National Research Foundation 12. Thompson M, Saag M, DeJesus E, Gathe J, Lalezari J, Landay AL,
Cade J, Enejosa J, Lefebvre E, Feinberg J. A 48-week randomized
(NRF), which is funded by the Korean government (MSIP). We
phase 2b study evaluating cenicriviroc versus efavirenz in treat-
thank Dr. Mi-Kyung Kim at Dong-A ST Co., Ltd. for technical
ment-naive HIV-infected adults with C-C chemokine receptor type
support and also thank professor Byung-Hyun Park at Chonbuk 5-tropic virus. AIDS. 2016;30:869-878.
National University Medical School for discussion. 13. Klibanov OM, Williams SH, Iler CA. Cenicriviroc, an orally active
CCR5 antagonist for the potential treatment of HIV infection. Curr
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Fischer L, Ratziu V. Efficacy and safety study of cenicriviroc for the
This study was sponsored by Dong-A ST Co., Ltd., Korea. treatment of non-alcoholic steatohepatitis in adult subjects with
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