Design and in vivo activity of A3 adenosine receptor agonist prodrugs

Article abstract of DOI:10.1007/s11302-020-09715-0

Prodrugs (MRS7422, MRS7476) of highly selective A₃ adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively, were synthesized by succinylation of the 2′ and 3′ hydroxyl groups, and the parent, active drug was shown to be readily liberated upon incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury model, 3?μmol/kg, p.o.), a known A₃AR effect. MRS7476 (5?mg/kg, p.o., twice daily) was found to protect against non-alcoholic steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 production, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2 levels) or inflammation.?Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls. Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body A₃AR knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables water solubility and in vivo activity of masked A₃AR agonists in models of two disease conditions.

Full text of DOI:10.1007/s11302-020-09715-0

Purinergic Signalling
https://doi.org/10.1007/s11302-020-09715-0
ORIGINAL ARTICLE
Design and in vivo activity of A3 adenosine receptor agonist prodrugs
R. Rama Suresh1 & Shanu Jain1 & Zhoumou Chen2,3 & Dilip K. Tosh1 & Yanling Ma4 & Maren C. Podszun4
Yaron Rotman4 & Daniela Salvemini2,3 & Kenneth A. Jacobson1
&
Received: 29 April 2020 /Accepted: 7 July 2020
#
This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2020
Abstract
Prodrugs (MRS7422, MRS7476) of highly selective A3 adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively,
were synthesized by succinylation of the 2′ and 3′ hydroxyl groups, and the parent, active drug was shown to be readily liberated upon
incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and
was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury
model, 3 μmol/kg, p.o.), a known A3AR effect. MRS7476 (5 mg/kg, p.o., twice daily) was found to protect against non-alcoholic
steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 produc-
tion, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2
levels) or inflammation. Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls.
Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body A3AR
knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables
water solubility and in vivo activity of masked A3AR agonists in models of two disease conditions.
.
.
.
.
.
Keywords Purinergic receptors Nucleosides Adenosine receptor Prodrug Pain Steatohepatitis
Abbreviations
ACTA2
ALT
AR
AUC
CCI
Chronic constriction injury
Smooth muscle α actin
Alanine transaminase
Adenosine receptor
Area under the curve
Cl-IB-MECA 2-Chloro-N6-(3-iodobenzyl)-adenosine-5′-N-
methyluronamide
G protein-coupled receptor
Hepatocellular carcinoma
GPCR
HCC
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11302-020-09715-0) contains supplementary
material, which is available to authorized users.
* Kenneth A. Jacobson
Yaron Rotman
rotmany@niddk.nih.gov
kennethj@niddk.nih.gov
R. Rama Suresh
rama.ravi@nih.gov
Daniela Salvemini
daniela.salvemini@health.slu.edu
Shanu Jain
shanu.jain@nih.gov
1
Laboratory of Bioorganic Chemistry, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health,
Bldg. 8A, Rm. B1A-19, 9000 Rockville Pike,
Zhoumou Chen
zhoumou.chen@health.slu.edu
Bethesda, MD 20892-0810, USA
2
3
4
Dilip K. Tosh
dilipkumar.tosh@nih.gov
Department of Pharmacology and Physiology, Saint Louis
University, St. Louis, MO, USA
Yanling Ma
mayl2007@126.com
Henry and Amelia Nasrallah Center for Neuroscience, Saint Louis
University, St. Louis, MO, USA
Maren C. Podszun
maren.podszun@gmail.com
Liver Diseases Branch, National Institute of Diabetes and Digestive
and Kidney Diseases, NIH, Bethesda, MD, USA

Purinergic Signalling
IB-MECA
N6-(3-iodobenzyl)-adenosine-5′-N-
methyluronamide
steatohepatitis (NASH) is underway, based on its modulation
of Wnt and other signaling pathways in liver tissue mediating
anti-inflammatory, anti-steatotic, and anti-fibrotic effects
[15–17]. Recently, the utility of highly selective agonists
chronic pain conditions was demonstrated [18–22]. These ag-
onists include MRS5698 3 and MRS5663 4, having a rigid,
extended substituent at the C2 position were shown to con-
tribute to high A3AR selectivity (thousands of fold) across
several species. The action of A3AR agonists in chronic neu-
ropathic pain models has been shown to occur in peripheral,
spinal, and supraspinal sites [18]. In a model of cancer
chemotherapy-induced neuropathic pain, MRS5698 modulat-
ed the nucleotide-binding oligomerization domain-like recep-
tor protein 3 (NLRP3) inflammasome, to attenuate proinflam-
matory IL-1β production and increase anti-inflammatory and
neuroprotective IL-10 expression [20].
IL-10
MRS5698
Interleukin 10
(1S,2R,3S,4R,5S)-4-(6-((3-chlorobenzyl)
amino)-2-((3,4-difluorophenyl)ethynyl)-9H-
purin-9-yl)-2,3-dihydroxy-N-methylbicyclo
[3.1.0]hexane-1-carboxamide
Non-alcoholic fatty liver disease
NAFLD activity score
Non-alcoholic steatohepatitis
Nucleotide-binding oligomerization domain-
like receptor protein 3 (inflammasome)
Oral glucose tolerance test
NAFLD
NAS
NASH
NLRP3
OGTT
SAR
TGFβ
Structure activity relationship
Transforming growth factor β
Many of the highly selective A3AR agonists display low
aqueous solubility, and we explore here, charged prodrug de-
rivatives of several agonists as a means of increasing solubility
while retaining in vivo activity. Drugs that have only sparing
aqueous solubility are subject to variability in their in vivo
responses [23]. Both peroral and parenteral drugs benefit from
sufficient solubility to avoid the use of complicated vehicles
for in vivo administration, which often drives the selection of
the most promising drug molecules. There are well-explored
approaches to derivatize nucleoside and nucleotide drugs as
prodrugs [23]. Often such prodrugs are intended for intracel-
lular action, for example, an O-acyl antiviral nucleoside
prodrug that is orally bioavailable, unmasked after entering
the cell, and phosphorylated for incorporation into viral
RNA [24]. However, nucleosides that target cell surface G
protein-coupled receptors (GPCRs) require a different
prodrug approach. Ester derivatives of nucleosides, specifical-
ly AR agonists [25], have also been reported to be prodrugs
Introduction
Adenosine acts as an anti-inflammatory mediator in many
physiological systems and is considered a retaliatory metabo-
lite; i.e., its production as a consequence of elevated ATP
inside and outside the cell is a protective feedback signal [1,
2]. Extracellular adenosine acts through a family of four G
protein-coupled receptors, two coupled principally to Gi (A1
and A3) [3, 4] and two coupled principally to Gs (A2A and
A
2B) [5, 6]. A3 adenosine receptor (A3AR) agonists IB-
MECA 1 and Cl-IB-MECA 2 (Fig. 1) have demonstrated
safety and efficacy in clinical trials for rheumatoid arthritis,
psoriasis (1), and hepatocellular carcinoma (HCC) and other
liver diseases (2) [7–11]. The A3AR is upregulated in patho-
logical conditions and can serve as a predictive biomarker for
clinical efficacy of A3AR agonists in inflammatory diseases
[12–14]. A clinical trial of Cl-IB-MECA in non-alcoholic
I
I
Fig. 1 Structures of selective
A3AR agonists in clinical trials (1,
2) and those used currently as
pharmacological probes (3, 4)
H
HN
HN
N
N
N
N
N
N
N
Cl
N
Cl
H
N
N
O
O
H3C
H3C
O
O
OH
OH
OH
OH
IB-MECA 1
Cl-IB-MECA 2
CH3
HN
Cl
HN
N
N
N
N
Cl
N
N
N
N
H
N
H
N
O
O
F
F
H3C
H3C
OH
OH
MRS5698 3
OH
OH
MRS5663 4

Purinergic Signalling
for unmasking in vivo, typically in the intestinal epithelium,
which is rich in esterases. Thus, we have chemically synthe-
sized O-succinyl derivatives of nucleosides 2 and 3, in which
the bioreversible esterification occurs at the free 2′- and 3′-
hydroxyl groups (Fig. 2A).
purchased from Sigma-Aldrich (St. Louis, MO). The prodrug
synthesis and characterization are described in the Supporting
information. H-NMR spectra were obtained with a Bruker
1
400 spectrometer using MeOH-d4 as a solvent. The chemical
shifts are expressed as parts per million, and the coupling
constants (J) are given in hertz. The RP-HPLC was performed
using Phenomenex Luna 5 μm C18(2) 100 Å, AXIA, 250 ×
21.2 mm column. The purity of final nucleoside derivatives
was checked using a Hewlett−Packard 1100 HPLC equipped
with an Agilent Eclipse 5 μm XDB-C18 analytical column
(50 mm × 4.6 mm; Agilent Technologies Inc., Palo Alto, CA).
Mobile phase: linear gradient solvent system, 10 mM TEAA
(triethylammonium acetate):CH3CN from 95:5 to 0:100 in
20 min; the flow rate was 1.0 mL/min. Peaks were detected
Methods
Instruments and reagents used for the synthesis
Cl-IB-MECA (2) was purchased from Tocris Bioscience
(Ellisville, MO). Compound 3 was synthesized in our labora-
tory as described.1,2 Other reagents and solvents were
Fig. 2 A Synthesis of the 2′,3′-di-
a
I
Cl
HN
N
HN
N
succinyl ester prodrugs using 8
equivalents of succinic anhydride
(a) in dichloromethane in the
presence of triethylamine (16
equivalents) and 4-
dimethylaminopyridine (1 equiv-
alent). The prodrugs were purified
to homogeneity by HPLC and
isolated as the partial Et3N salt
forms (0.8 equivalents Et3N for
MRS7422, 0.5 equivalents for
MRS7476). B Enzymatic hydro-
lysis of MRS7422 using porcine
liver esterase (PLE, b) at 37 °C in
HEPES buffer, pH 7.3. Mean
SD is shown. See Figure S3 for
the corresponding data for
N
N
N
N
N
N
Cl
H
H
N
N
O
O
F
F
H3C
H3C
O
OH
OH
Cl-IB-MECA 2
OH
OH
MRS5698 3
a
a
b
b
I
Cl
HN
HN
N
N
N
N
N
N
N
Cl
N
H
N
H
N
MRS7476
O
O
O
F
F
H3C
H3C
O
O
O
O
O
O
O
O
CO2H
CO2H
CO2H
CO2H
MRS7422 5
MRS7476 6
b
120
100
80
60
40
20
0
0
2
4
6
24
48
72
Hours
MRS 7422
2-Cl-IB-MECA

Purinergic Signalling
by UVabsorption with a diode array detector at 230, 254, and
280 nm. All derivatives tested for biological activity showed
> 95% purity in the HPLC systems. cLogP was calculated
using ChemDraw 19.1 (PerkinElmer) for MAC.
Mouse maintenance and diet
A3AR knockout (A3Δ/Δ) and WT (A3+/+) mice, on a C57BL/
6 J background, used in this study were generated as de-
scribed.27 Briefly, mice heterozygous for A3AR (A3Δ/+) were
interbred to generate A3Δ/Δ and WT mice. Male littermates
were used for all experiments.
Expression of adenosine receptors in human liver
Mice were housed on a 12-h light/12-h dark cycle in a
pathogen-free barrier facility at room temperature (23 °C).
The mice had ad libitum access to water. The mice were main-
tained on standard mouse chow (7022 NIH-07 diet, 15% kcal
fat, energy density 3.1 kcal/g, Envigo, Inc., Huntingdon, UK)
or a high fat diet (HFD, 36% fat, equivalent to 60% kcal; 36%
carbohydrate; F3282; Bio-Serv, Flemington, NJ). Mice main-
tained on chow diet (CD) were transferred to HFD at 13 weeks
of age, and mice were compared after 32 weeks on HFD. The
study and its procedures were approved by the Animal Care
and Use Committee of National Institute of Diabetes and
Digestive and Kidney Diseases, animal protocol K083-LBC-
17.
Gene expression of ARs was assessed in baseline (pre-
treatment) liver biopsies obtained in a clinical trial investigat-
ing the effects of vitamin E in patients with non-alcoholic fatty
liver disease (ClinicalTrials.gov, NCT017921150) [reference
currently under review]). The study was reviewed by the
Institutional Review Board, and all subjects gave written
informed consent. Liver samples were placed in
RNAlater (Thermo Fisher Scientific, Waltham, MA) at
the bedside and stored in − 80 °C. RNA was extracted
using Trizol Reagent (Thermo Fisher Scientific) followed
by purification with miRNAeasy kit (Qiagen, Hilden,
Germany). The quality of extracted RNA was assessed
using a NanoDrop spectrophotometer and Agilent 2100
Bioanalyzer. PolyA-enriched RNA was used to prepare a
paired-end index library. Samples were pooled (2 pools)
and run on an Illumina HiSeq4000 with 75 bp paired-end
sequencing. Reads were aligned to the human genome
(GRCh38) using STAR version 2.5.4a with default set-
tings. Genes were counted with htseq-count using the
mode intersection-nonempty. Counts were normalized
and are shown as transcripts per million (TPM). Data were
compared with normal liver tissue (n = 10) from the HPA
data set obtained through the Human Protein Atlas (https://
www.proteinatlas.org, PMID: 25613900).
Gene expression analysis by quantitative real time-
PCR
Gene expression analysis was carried out as described [27].
Liver tissues were dissected and were frozen in liquid nitro-
gen. RNAeasy mini kit (Qiagen, Germantown, MD) was used
for the extraction of total liver mRNA. After synthesizing
cDNA using Superscript III First Strand synthesis Super
Mix (Invitrogen, Thermo Fisher, Rockville, MD), qRT-PCR
was performed using SYBR green reagent (Applied
Biosystems, Beverly, MA) in triplicates. 18s rRNA was used
as the normalization control for relative gene expression anal-
ysis using ΔΔCt method. Primer sequences are provided in
Table S2.
Expression of Adora3 in mouse liver cells
Total RNA was extracted from primary hepatocytes and stel-
late cells isolated from C57BL/6 mice. First-strand cDNAwas
used as template for TaqMan probe-based quantitative poly-
merase chain reaction (qPCR) assay to quantify the gene ex-
pression level, which was normalized to the level of 18S
rRNA. Primer sequences are in Table S1 (Supporting
Information).
Histological assessment of liver sections from HFD
A3Δ/Δ mice
Liver tissues were dissected out of A3Δ/Δ and WT mice after
32 weeks on HFD and were frozen in liquid nitrogen. Liver
tissues were sectioned and stained using standard techniques.
Hematoxylin-eosin (HE) stained liver sections were visual-
ized using Keyence Microscope BZ-9000.
Mouse model of chronic neuropathic pain
The mouse experimental model of chronic neuropathic pain
(CCI model [26]) has been published in various studies show-
ing protection by highly selective A3AR agonists [19, 21].
Briefly, the drugs were administered p.o. on day 7 following
constriction of the sciatic nerve, which is the time of peak
pain. Following drug injection, mechano-allodynia was quan-
tified at multiple time points according to the hind paw with-
drawal response to von Frey filaments stimulation.
Statistics
All data are expressed as the mean SEM. Data were tested
for statistical significance by two-tailed, unpaired Student’s t
test, as appropriate. A P value of less than 0.05 was considered
significant.

Purinergic Signalling
Results
publication of Besada et al. [25] and other studies of SAR
[4], in which acylation of both 2′- and 3′-hydroxyl groups
greatly reduces or precludes affinity at ARs. This is also con-
sistent with the steric crowding in the now well-characterized
ribose binding site in the A2AAR (which is structurally anal-
ogous in other AR subtypes), as the 2′- and 3′-hydroxyl
groups are required for H-bond formation with polar groups
of the receptor [4]. The cLogP values for the prodrugs com-
pared with the active drugs were − 6.11 (−0.187 as neutral
acid) and − 4.02 (1.90 as neutral acid) for 5 and 6, respective-
ly, compared with − 1.42 and 1.54 for 2 and 3, respectively.
However, experimental LogP data were not determined for
this class of compounds. The enhancement of aqueous solu-
bility with this derivative was particularly pronounced. The
maximum solubility of MRS7476 in water was 2.5 mg/ml
(3.1 mM), which is > 1000-fold greater than the aqueous sol-
ubility of MRS5698 [19]. The maximum solubility of
MRS7422 in water was 3.0 mg/ml (3.6 mM), potential off-
target activities at 46 receptors were measured by the
Psychoactive Drug Screening Program, and only μM affinity
was determined for several sites (Table S1, Supporting
Information) [34].
Prodrug design and synthesis
The recent generation of highly specific A3AR agonists, such
as MRS5698, generally has low aqueous solubility. The max-
imal solubility of MRS5698 was determined to be only
1.6 μg/ml at pH 7.4, consistent with its bearing hydrophobic
substituents at C2 and N6 positions [19]. In comparing
prodrug strategies used with nucleosides, we did not want to
use a derivatization scheme that would increase diffusion into
the cell because of the cell surface location of the A3AR.
Rather, we targeted liberation of the free nucleoside by ester
formation, that is subject to hydrolysis by widespread ester-
ases, including in the intestinal epithelium. Simple alkyl ester
prodrugs of various A3AR nucleoside antagonists and ago-
nists, including Cl-IB-MECA, were previously reported to
require hydrolysis in order to liberate significant pharmaco-
logical activity associated with the parent drugs [25]. Small 2′
and 3′ alkyl diesters were found to be more readily cleaved
enzymatically than similar diesters with large alkyl groups,
such as 2′,3′-dihexanoate esters. Since the free ribosyl hydrox-
yl groups of Cl-IB-MECA and other nucleoside agonists are
generally needed for AR activation [4], we again planned to
block these groups in prodrug derivatives. None of the previ-
ously reported ester prodrugs of A3AR ligands [25] utilized
water-solubilizing succinyl esters, although this has been
attempted for other prodrugs, linkers, and nanocarrier assem-
blies [28–31]. The approach of adding a water-solubilizing
group to form prodrugs of poorly soluble parent drugs is often
accomplished with a phosphate ester [32]. However, we were
unable to synthesize and purify analogous 2′ or 3′-phosphate
esters of Cl-IB-MECA.
The two prodrugs were first tested for the ability to be
cleaved by porcine liver esterases. Under the incubation con-
ditions used (37 °C, pH 7.3, 72 h), both were converted to the
corresponding parent drug as determined by HPLC, but
MRS7422 (Fig. 2B) was more rapidly cleaved than
MRS7476 (Figure S3, Supporting information). Thus, we ex-
pect that these prodrugs will lead to the generation of the
active drug molecule in vivo.
Activity in a chronic pain model
These two A3AR agonists, Cl-IB-MECA (already in clin-
ical trials for liver conditions) and MRS5698 (a pharmacolog-
ical probe compound), were derivatized as 2′,3′-di-succinyl
monoesters, which were isolated as triethylammonium salt
forms. The synthetic method is straightforward (Fig. 2A,
with details in the Supporting information): treatment of the
parent nucleoside with excess succinic anhydride in dichloro-
methane in the presence of triethylamine and acylation cata-
lyst 4-(dimethylamino)pyridine. Each of the prodrug mole-
cules, MRS7422 5 and MRS7476 6, was purified by HPLC
and lyophilized as a salt form. The structures were confirmed
by NMR and mass spectrometry (Figure S2, Supporting
information), and partial equivalents of triethylamine were
associated with each. As anticipated, the positions of deriva-
tization were shown by NMR to be at the 2′- and 3′-hydroxyl
groups.
To determine the in vivo efficacy of the novel prodrugs we
tested them first in an acute-administration setting using the
chronic constriction injury (CCI) mouse model [26], where a
single dose of an A3AR agonists can reverse established
mechano-allodynia at day 7 post-injury (time of maximal
pain), and its efficacy and duration are measurable in real time.
The two prodrugs were administered by oral gavage (1 or
3 μmol/kg) in mice in the CCI model, as reported in our
previous structure activity relationship (SAR) studies [19,
21]. This allowed the real-time in vivo efficacy of the A3AR
agonists at the ipsilateral (nerve injured) paw to be followed.
The parent drug Cl-IB-MECA was compared directly to its
prodrug MRS7422, and the activity of the prodrug reached a
higher efficacy at the same dose (Fig. 3A). However, the effect
of MRS7422 at 1 μmol/kg was relatively short lasting. In
contrast, the effect of MRS7476 at 3 μmol/kg was still signif-
icant at 4-h post-administration (Fig. 3B), which is similar to
the time course of pain protection by the parent drug
MRS5698 [19]. There was no effect of either nucleoside de-
rivative at the contralateral paw.
The reported A3AR affinity (Ki, nM) is 1.5 (human) and
0.80 (mouse) for Cl-IB-MECA and 3.49 (human) and 3.08
(mouse) for MRS5698 [22, 33]. We did not subject the
prodrugs to AR binding studies, based on our earlier

Purinergic Signalling
a
a
b
4
3
2
1
0
2-Cl-IBMECA (1 mol/kg)
2-Cl-IBMECA (3 mol/kg)
MRS7422 (1 mol/kg)
MRS7422 (3 mol/kg)
MRS7476 (3
oral
m
ol/kg)
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
oral
Day 0
0
1
2
3
4 5
Day 0
0
1
2
3
4 5
Time (h) post
treatment on Day 7
Time (h) post
treatment on Day 7
ADORA1 ADORA2A ADORA2B ADORA3
b
3
2
1
0
Fig. 3 Anti-hyperalgesia effects of orally administered prodrugs in the
mouse CCI model. A Effects of Cl-IB-MECA at 1 μmol/kg (n = 5) com-
pared with MRS7422 at 1 μmol/kg (n = 2) and 3 μmol/kg (n = 3). B
Effects of MRS7476 at 3 μmol/kg (n = 2)
p=0.02
Given its apparent benefit in the CCI model, it was decided
to apply MRS7476 as the prodrug analogue to be used when
measuring the response to chronic administration in a model
of NASH. However, prior to testing this A3AR agonist
prodrug in a mouse therapeutic experiment in a NASH model,
we tested and found Adora3 to be expressed in the mouse
liver. Specifically, we measured the expression of the receptor
in liver tissue and cells. All four ARs were expressed in hu-
man liver tissue from NAFLD patients (n = 19; Fig. 4A).
When comparing hepatic ADORA3 expression between pa-
tients with NAFLD and controls with normal liver (n = 10),
we found a 1.9-fold decreased expression in the NAFLD pa-
tients (Fig. 4B; p = 0.02). To determine which cells in the liver
predominantly express the receptor, we compared its expres-
sion between primary mouse hepatocytes and stellate cells, the
main drivers of hepatic fibrosis. The expression level of
Adora3 was 27.6-fold greater in the stellate cells (p = 0.008;
Fig. 4C).
NAFLD
Healthy
c
50
40
30
20
10
p=0.008
4
2
0
Hepatocytes
Hepatic
Stellate Cells
Fig. 4 A mRNA expression of all four ARs in liver tissue from human
NAFLD patients by RNA sequencing. B Comparative expression of
ADORA3 in liver tissue from NAFLD patients and controls with normal
liver. C Expression of ADORA3 by qPCR in primary mouse hepatocytes
and stellate cells. NAFLD, non-alcoholic fatty liver disease. TPM tran-
scripts per million
Activity in a model of NASH
The mouse STAM NASH model [37–39] was used to test the
efficacy of MRS7476. NASH was established in male
C57BL/6 mice by a single subcutaneous injection of 200 μg
streptozotocin 2 days after birth and feeding with a high fat
diet ad libitum after 4 weeks of age (original study plan in
Figure S4, Supporting Information). Three groups of mice
were examined (treatment beginning at 6 weeks of age, for
3 weeks): vehicle control, MRS7476, and telmisartan (angio-
tensin II receptor antagonist, 10 mg/kg, p.o., once daily, as a
positive control) [40]. The impact of chronic drug treatment
on liver histology (Fig. 5A) and other markers were examined
(Tables S4–S6 and protocol details in the Supporting
Information). MRS7476 (5 mg/kg, p.o., twice daily) had no
effect on hepatic triglycerides (Fig. 6C) or histological
steatosis scores (Fig. 7A). Similarly, no effect was seen on
histological inflammation (Fig. 7B) scores or serum ALT
(Fig. 6A), but markedly improved hepatocyte ballooning
(Fig. 7C), the hallmark of NASH-associated hepatocyte inju-
ry, was observed. The composite NAFLD activity score
(NAS) was significantly improved by MRS7476 (Fig. 5B).
MRS7476 did not affect liver fibrosis.
There was no impact of treatment on hepatic TGFβ and
ACTA2 expression [35, 36]. Plasma IL-10 levels were in-
creased in MRS7476-treated animals (Fig. 6B; p = 0.01).
Two of 6 animals treated with MRS7476 (and none treated
with vehicle) developed significant weight loss starting at day
10 and 14 of treatment and were found dead on day 17 and 18,
respectively (Figure S5, Supporting Information). Notably,
these two mice had markedly lower baseline body weights
compared with the other animals and to the control group.
The cause for excess mortality is not clear, and the dead ani-
mals were not included in analyses. The weight drop on the

Purinergic Signalling
32 weeks. mRNA levels of inflammatory markers in liver
tissue of A3Δ/Δ mice and WT mice maintained on HFD were
examined by RT-PCR. Monocyte chemoattractant protein 1
(Mcp1) mRNA levels were increased in the liver of A3Δ/Δ
mice (Fig. 8). Accordingly, the gene expression levels of mac-
rophage marker, cluster of differentiation 68 (Cd68), was in-
creased in the liver of A3Δ/Δ mice. Whole body knockout of
A3AR also resulted in significant increase in mRNA levels of
macrophage inflammatory protein-1 alpha (Mip1a) in liver
tissue (Fig. 8). Gene expression of pro-inflammatory cyto-
kines such as interleukin 6 (Il6) and tumor necrosis factor
alpha (Tnfa) was increased in the liver of A3Δ/Δ mice (Fig.
8). No differences were observed in mRNA levels of genes
coding for interferon-gamma (Ifng), interleukin 1-alpha (Il1a),
interleukin 1-beta (Il1b), and macrophage inflammatory
protein-1 (Mip1b) (Fig. 8). Our data suggest enhanced expres-
sion of inflammation markers in livers of mice lacking func-
tional A3AR. Further, liver tissues from HFD A3Δ/Δ mice and
WT mice were sectioned and HE stained. Thus, the lack of
A3AR may enhance the severity of liver steatosis and second-
ary hepatic inflammation associated with obesogenic diet.
Discussion
The objective was to demonstrate that we could reversibly
mask, in a highly water-soluble form, known A3AR agonists
using a common chemical approach that potentially applies to
both riboside agonists and (N)-methanocarba derivatives. One
of the prodrugs was tested by oral administration in two
in vivo mouse models to demonstrate its action in two known
activities of A3AR agonists, i.e., anti-hyperaglesia in the
chronic pain state and protection in NASH. We showed that
the otherwise poorly soluble parent drug, MRS5698, was
formed by the action of esterases, such as are plentiful
in vivo. In vitro, MRS7422 was more readily hydrolyzed en-
zymatically by liver esterase in vitro compared with the rigid
bicyclic methanocarba derivative MRS7476. We did not de-
termine the rate of cleavage by intestinal or gastric epithelial
esterases, but these tissues are likely sites for hydrolysis and
absorption, by analogy to a published study of such esterases
acting on chlorogenic acid esters present in coffee [41]. Also,
the rate of hydrolysis within mouse tissues leading to the real
time reversal of chronic neuropathic pain within 1 h cannot be
compared quantitatively to the slower kinetics in the model
in vitro study of PLE hydrolysis of MRS7476.
Fig. 5 Histological effects in the liver in STAM mice following
administration of MRS7476 (5 mg/kg, p.o., twice daily, for 3 weeks) or
telmisartan included as a positive control. Data corrected using the
Bonferroni multiple comparison test
last day in the other animals was likely due to the blood vol-
ume required for the OGTT test (~1% of body weight), rather
than an A3 agonist-related effect, as the safety of chronically
administered A3 agonists has been demonstrated [7–9, 11]..
Enhanced hepatic inflammation and steatosis in A3Δ/Δ
mice fed HFD
Obesity is associated with enhanced risk of developing non-
alcoholic fatty liver disease (NAFLD). Development of liver
steatosis and secondary chronic liver inflammation are key
factors contributing to metabolic disorders. To examine the
effect of the A3AR on the development of liver steatosis and
inflammation associated with obesity, we subjected control
and A3AR knockout mouse lines to HFD for the period of
NASH is a progressive disease often associated with obe-
sity that can lead to cirrhosis and liver cancer, and its incidence
is rapidly increasing [42]. Thus, the cost to society will be
enormous, and novel, effective pharmacological treatments
are needed. Drugs in clinical trials were recently reviewed
[43]. NASH patients typically display histological liver pa-
thology, such as cytoskeletal damage (hepatocyte ballooning),

Purinergic Signalling
Fig. 6 Biochemical measures of
damage in the liver of STAM
mice following administration of
MRS7476 (5 mg/kg, p.o., twice
daily, for 3 weeks) or telmisartan
included as a positive control.
Statistics used the Bonferroni
multiple comparison test
steatosis, and inflammation. Fibrosis is often associated with
the later stages of the condition. The chemically induced
STAM model has become a standard in the field of drug de-
velopment for NASH treatment [44, 45]. In a study of the
effects of the parent drug Cl-IB-MECA in the STAM model,
NASH was decreased with reduction of both inflammatory
and steatotic parameters [15]. In addition, use of the same
agonist in the carbon tetrachloride model, improved liver
Fig. 7 Scoring of liver damage in
STAM mice. The A3AR agonist
prodrug MRS7476 was compared
with vehicle alone or telmisartan
included as a positive control

Purinergic Signalling
Fig. 8 Enhanced inflammation
and steatosis in the liver of HFD
A3Δ/Δ mice. A Real-time PCR
analysis of genes coding for he-
patic inflammatory markers. 18s
RNA was taken as a normaliza-
tion control. All data are given as
mean + SEM with *p < 0.05
using two-tailed, unpaired
Student’s t test (n = 3–5, each
group). B HE Stain of liver sec-
tions from WT and A3Δ/Δ mice
kept on HFD. Microscopic anal-
ysis using × 20 magnification
inflammation and fibrosis and normalized alanine aminotrans-
ferase (ALT), to clearly demonstrate an anti-NASH effect of a
selective A3AR agonist [15]. One of the A3AR agonist
prodrugs synthesized here, MRS7476, was active in vivo in
improving NASH. We have shown here that hepatic stellate
cells, the main effectors of hepatic fibrosis, have much higher
expression levels of mRNA encoding the A3AR than hepato-
cytes. We did not establish if these expression levels are al-
tered in the NASH mouse model or in A3AR agonist-treated
mice.
In the STAM model of NASH, treatment with MRS7476
showed a significant improvement in the NAFLD activity score
(NAS), driven mainly by a remarkable reduction in hepatocyte
ballooning. In addition, plasma IL-10 level was increased in the
MRS7476 group compared with the vehicle group. This result
suggests that MRS7476 has anti-NASH potential in this model.
We have not determined whether the prodrug derivatization of
MRS5698 led to its lack of hepatoprotective effects on liver
enzymes and reduced fibrosis in this NASH model, as was seen
with another A3AR agonist, Cl-IB-MECA [15]. Further char-
acterization including in vivo pharmacodynamic analysis of
prodrug MRS7476 would be appropriate.
unmasking in vivo, in disease models already associated with
beneficial A3AR action. We have shown that both ester bonds
on each molecule are cleaved by liver esterase in vitro, and by
deduction in vivo. Of the two compounds prepared, the
riboside MRS7422 was more readily hydrolyzed enzymatical-
ly in vitro compared with the rigid bicyclic methanocarba
derivative MRS7476. We then subjected this prodrug ap-
proach to models of two disease conditions, in which the drug
was administered orally to simulate this treatment modality for
chronic human diseases. However, the longer duration of pro-
tection by MRS7476 compared with MRS7422 in a real-time
in vivo assay of mechano-allodynia prompted us to examine
the methanocarba derivative MRS7476 in a test of protection
against NASH [46], and a benefit was observed. The unusu-
ally high selectivity of the parent drug MRS5698 offers con-
fidence that A3AR is responsible, along with evidence of re-
ceptor expression in liver stellate cells and a gene expression
pattern consistent with increased injury in A3AR knockout
mouse liver. Selective A3AR agonists are already in clinical
trials for inflammatory diseases (IB-MECA) and liver diseases
(Cl-IB-MECA), including NASH, and are proposed for the
future treatment of chronic pain. Thus, we have expanded
the toolbox of useful nucleoside derivatives for studying these
and other actions of A3AR agonists in vivo.
Conclusions
Funding information This work has been supported by funds from SLU
(Salvemini Start-Up Funds) and NIDDK Intramural Research Program
(Jacobson, ZIADK31117; Rotman, ZIADK75013).
We have introduced 2′,3′-di-succinylation of potent and selec-
tive A3AR agonists as a reversible prodrug strategy. This ap-
proach greatly increased water solubility in an otherwise dif-
ficult to dissolve compound and allowed us to observe salu-
tary activity of masked A3AR agonists, i.e., prodrugs for
Compliance with ethical standards
Conflict of interest All other authors claim no conflicts of interest.

Purinergic Signalling
Ethical approval All procedures were conducted in accordance with the
International Association for the Study of Pain, the National Institutes of
Health guidelines on laboratory animal welfare guidelines, and the ap-
proval of Saint Louis University and NIDDK Institutional Animal Care
and Use Committees.
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