Characterization of the properties of a selective, orally bioavailable autotaxin inhibitor in preclinical models of advanced stages of liver fibrosis

Article abstract of DOI:10.1111/bph.14118

Background and Purpose: Autotaxin (ATX) is a secreted phospholipase which hydrolyses lysophosphatidylcholine to generate lysophosphatidic acid (LPA). The extracellular signalling molecule LPA exerts its biological actions through activation of six GPCRs expressed in various cell types including fibroblasts. Multiple preclinical studies using knockout animals, LPA receptor antagonists or ATX inhibitors have provided evidence for a potential role of the ATX/LPA axis in tissue fibrosis. Despite growing evidence for a correlation between ATX levels and the degree of fibrosis in chronic liver diseases, including viral hepatitis and hepatocellular carcinoma, the role of ATX in non-alcoholic steatohepatitis (NASH) remains unclear. Experimental Approach: The relevance of ATX in the pathogenesis of liver fibrosis was investigated by oral administration of Ex_31, a selective ATX inhibitor, in a 10?week model of carbon tetrachloride-induced liver injury and in a 14?week model of choline-deficient amino acid-defined diet-induced liver injury in rats. Key Results: Oral administration of Ex_31, a selective ATX inhibitor, at 15?mg·kg^?1 twice daily in therapeutic intervention mode resulted in efficient ATX inhibition and more than 95% reduction in plasma LPA levels in both studies. Treatment with Ex_31 had no effect on biomarkers of liver function, inflammation, or fibrosis and did not result in histological improvements in diseased animals. Conclusions and Implications: Our findings question the role of ATX in the pathogenesis of hepatic fibrosis and the potential of small molecule ATX inhibitors for the treatment of patients with NASH and advanced stages of liver fibrosis.

Full text of DOI:10.1111/bph.14118

Title: Characterisation of the properties of a selective, orally bioavailable autotaxin
inhibitor in preclinical models of advanced stages of liver fibrosis
Running title: Autotaxin inhibition in models of liver fibrosis
Authors: Manuel Baader, Tom Bretschneider, Andre Broermann, Joerg F. Rippmann, Birgit
Stierstorfer, Christian A. Kuttruff, Michael Mark
Author affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65,
88397 Biberach an der Riss, Germany
Corresponding author: Manuel.Baader@boehringer-ingelheim.com
Abstract
Background and Purpose: Autotaxin (ATX) is a secreted phospholipase which hydrolyses
lysophosphatidylcholine (LPC) to generate lysophosphatidic acid (LPA). The extracellular
signalling molecule LPA exerts its biological actions through activation of six G protein-
coupled receptors expressed in various cell types including fibroblasts. Multiple preclinical
studies using knockout animals, LPA receptor antagonists or ATX inhibitors have provided
evidence for a potential role of the ATX/LPA axis in tissue fibrosis. Despite growing
evidence for a correlation between ATX levels and the degree of fibrosis in chronic liver
diseases, including viral hepatitis and hepatocellular carcinoma, the role of ATX in non-
alcoholic steatohepatitis (NASH) remains unclear.
Experimental Approach: The relevance of ATX in the pathogenesis of liver fibrosis was
investigated by oral administration of Ex_31, a selective ATX inhibitor, in a 10-week model
of carbon tetrachloride (CCl4)-induced liver injury and in a 14-week model of choline-
deficient amino acid–defined diet (CDAA) induced liver injury in rats.
Key Results: Oral administration of Ex_31, a selective ATX inhibitor, at 15 mg  kg-1 twice
daily in therapeutic intervention mode resulted in efficient ATX inhibition and more than 95%
reduction in plasma LPA levels in both studies. Treatment with Ex_31 had no effect on
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doi: 10.1111/bph.14118
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biomarkers of liver function, inflammation or fibrosis and did not result in histological
improvements in diseased animals.
Conclusions and Implications: Our findings question the role of ATX in the pathogenesis of
hepatic fibrosis and the potential of small molecule ATX inhibitors for the treatment of
patients with NASH and advanced stages of liver fibrosis.
Abbreviations
ACTA2 alpha smooth muscle actin gene
ALT
alanine aminotransferase
ATX
autotaxin
CCL2
CCl4
CC-chemokine ligand 2
carbon tetrachloride
CDAA
CSAA
CTGF
Ctrl
choline-deficient amino acid-defined
choline-supplemented amino acid-defined
connective tissue growth factor
control
CXCL1
C-X-C motif chemokine ligand 1
epidermal growth factor-like module-containing mucin-like hormone receptor-
EMR1
like 1
Ex_31
FCS
h
Example 31(24)
fetal calf serum
hours
H&E
HSC
haematoxylin and eosin stain
hepatic stellate cell
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HYP
IC50
hydroxyproline
drug concentration resulting in 50% inhibition of control activity
ITGAM integrin alpha M
LPA
lysophosphatidic acid
LPAR
LPC
lysophosphatidic acid receptor
lysophosphtidylcholine
Masson's trichrome stain
minutes
Masson
min
MS
mass spectrometry
NAS
NASH
non-alcoholic steatohepatitis activity score
non-alcoholic steatohepatitis
RT-PCR reverse transcription polymerase chain reaction
SD
standard deviation
SEM
standard error of mean
TGFB
TNFA
TRAIL
transforming growth factor beta
tumor necrosis factor alpha
tumor necrosis factor-related apoptosis-inducing ligand
SMA alpha smooth muscle actin
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Introduction
Autotaxin (ATX) is a secreted enzyme of the phospholipase superfamily which hydrolyses
lysophospholipids to generate lysophosphatidic acid (LPA) (Perrakis & Moolenaar, 2014).
Mice deficient in ATX die during embryonic development, whereas heterozygous mice
survive to adulthood and display plasma LPA levels that are approximately half that of wild-
type mice, implying that ATX is the major source of plasma LPA in mice (Tanaka et al.,
2006; van Meeteren et al., 2006). Recently, it was demonstrated that anti-ATX DNA aptamers
block LPA production by more than 90% in human serum, demonstrating that ATX is a major
source of LPA in human plasma (Kato et al., 2016). Lysophosphatidic acid acts as an
extracellular signalling molecule and exerts its biological actions through the activation of G
protein-coupled receptors (GPCRs). To date, six GPCRs have been described that are
involved in LPA signalling (LPA1 receptor (LPAR1), LPAR2, LPAR3, LPAR4, LPAR5,
LPAR6) and each is coupled to various signalling cascades including G12/13, G , G and
q
s
G (Kihara et al., 2014). Elevated plasma LPA concentrations and increased LPAR
I
expression have been observed in patients suffering from chronic inflammatory diseases,
fibrosis and cancer (reviewed in (Chu et al., 2015; Leblanc & Peyruchaud, 2015; Sevastou et
al., 2013). Associations between LPAR signalling and various diseases have stimulated
interest within the pharmaceutical industry for the development of LPAR antagonists (Llona-
Minguez, Ghassemian & Helleday, 2015). However, it has proven challenging to identify
potent and selective LPAR antagonists with few compounds having been advanced to clinical
development (Khanna et al., 2014; Pasquinelli, 2013).
Autotaxin as the major source of plasma LPA has attracted the interest of researchers and the
pharmaceutical industry. Several small molecule ATX inhibitors have been developed for use
in various inflammatory and fibrotic diseases (reviewed in (Barbayianni et al., 2015;
Castagna et al., 2016)). Of the candidates, GLPG1690 is currently being investigated in a
Phase II trial in patients with idiopathic pulmonary fibrosis. Beyond that, there is growing
evidence to support the involvement of ATX in the pathogenesis of liver fibrosis related to
viral hepatitis and hepatocellular carcinoma (Kondo et al., 2014; Nakagawa et al., 2011; Pleli
et al., 2014). However, the role of ATX in non-alcoholic steatohepatitis (NASH) remains
unclear.
The elevated ATX and LPA levels in patients with chronic liver diseases from different
etiologies have prompted efforts to explore the role of the LPA/ATX axis in the pathogenesis
of NASH-related fibrosis. The aim of the present study was to investigate the effect of LPA
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in primary human hepatic stellate cells (HSC) in vitro and to characterise the properties of
Ex_31, a selective small molecule ATX inhibitor, in preclinical models of advanced liver
fibrosis.
Methods
Synthesis and purification of Ex_31
The ATX inhibitor Ex_31 (Example 31(24)) was synthesized according to the procedures
described in patent WO 2012/005227 and EP 2592081 A1 respectively (Supplemental Figure
S9). The synthesis of Ex_31 started from commercially available 7-azaindole S1, which upon
treatment with dimethylamine and formaldehyde underwent a Mannich reaction to afford 7-
azagramine S2. The corresponding nitroethyl-derivative S3 was next obtained through
reaction with nitromethane and dimethylsulfate under basic conditions. Subsequently, the
nitro group present in S3 was converted to the amine S4 through hydrogenation using
Pearlman’s catalyst. In order to construct the tricyclic core present in S5, the aza-tryptamine
S4 was heated with formaldehyde under acidic conditions in a sealed tube which triggered the
desired Pictet-Spengler cyclization to yield S5. Boc-protection of S5 afforded S6, the latter
which was alkylated with 1-(bromomethyl)-4-chloro-2-fluorobenzene under basic conditions
to afford S7. Deprotection of the Boc group was achieved under acidic conditions and yielded
the tricyclic amine S8. The latter was coupled with carboxylic acid S9 (synthesized in 3 steps
from commercially available trans-1,4-cyclohexanedicarboxylic acid monomethyl ester
according to procedures described in patent WO 2012/005227 and EP 2592081 A1
respectively) using HOBt and EDC to yield an amide which was subsequently subjected to
ester hydrolysis to afford the final carboxylic acid Ex_31. The final crude compound was
purified by silica gel column chromatography (eluent: 1% MeOH:dichloromethane) to
provide Ex_31 as a white solid.
Primary human hepatic stellate cell culture
Primary human HSCs (Innoprot, Spain) were maintained in humidified cell culture incubators
at 37°C and 5% CO2. Cells were cultured in standard cell culture plastic ware according to
manufacturer’s instructions. Cells were grown in SteCM medium (ScienCell, USA)
containing growth supplements and fetal calf serum (FCS) (2%). Cells were split when they
reached sub-confluence.
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Real–time polymerase chain reaction analysis of gene expression in primary human
hepatic stellate cells
For gene expression studies, HSCs were seeded in 12-well plates and serum starved for 18 h
before treatment with LPA (1-Oleoyl LPA, 10 M 18:1; Cayman Chemicals, USA) for 1 h,
2 h, 4 h and 6. Cells were lysed in RLT-buffer and total RNA was isolated using RNeasy
Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Concentrations of
RNA were analysed with a NanoDrop® ND-1000 UV-Vis spectrophotometer at 260 nm
(Thermo Fisher Scientific, USA). Purity of RNA was confirmed using 260/280 nm ratios
(pure samples show a range of 1.8 – 2.1) and samples were stored at -80°C before further
processing. For reverse transcription PCR (RT-PCR), RNA (50 – 2000 ng) was
reverse-transcribed into cDNA with a high capacity cDNA archive kit (Thermo Fisher
Scientific) in accordance with the manufacturer’s instructions, and cDNAs were stored at -
20°C. For the analysis of gene-specific mRNA expression, cDNA was amplified by real–time
PCR with specific primers and fluorescently labelled probes (Supplemental Table S8) using a
thermal cycler (Eppendorf, Germany). Results were evaluated using SDS software
version 2.2 (Thermo Fisher Scientific). Expression levels were normalised in reference to the
house keeping gene RNA polymerase 2. For calculation of relative changes in gene
expression, values of individual samples were divided by the mean value of untreated
samples at time zero.
Measurement of TRAIL-induced hepatic stellate cell apoptosis
For the measurement of tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL)-induced apoptosis, white opaque 384 well plates were seeded with HSCs at a
density of 2,000 cells per well in SteCM medium supplemented with FCS (2%) and growth
supplements (1%). Six hours later, medium was replaced by SteCM medium without
supplements and cells were serum-starved for 6 h. For the apoptosis assay, cells were
stimulated for 24 h with different concentrations of LPA alone or in combination with TRAIL
(30 ng  mL-1; R&D Systems, Germany). Caspase 3/7 activity was measured using a
luminescence based Caspase-Glo 3/7 assay system (Promega, USA) according to the
manufacturer’s instructions.
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In vitro ADME assays
Plasma protein binding (PPB)
The plasma protein binding was determined by equilibrium dialysis. Teflon dialysis cells
with a cutoff of 5 – 10 kDa were used. Ethylendiamintetraessigsäure (EDTA) plasma was
spiked with 10 mM Ex_31 and transferred to the donor chamber. The acceptor chamber was
filled with phosphate buffered saline (PBS) buffer (pH 7.4) supplemented with dextran. The
chamber was incubated for 3 h under rotation at 37 °C. Aliquots from both chambers were
collected and analysed by LC-MS/MS for calculation of the PPB.
The following assays were performed as described previously (Luippold et al, 2011):
Cytochrome P450 assay
Cytochrom P450 oxigenase specific substrates were incubated with Ex_31 at 37 °C with liver
microsomes. The assay was performed in 0.1 M Tris buffer supplemented with 5 mM MgCl2
and 1 mM NADPH. Ex_31 was tested in a range of 0 – 50 µM.
Caco-2 assay
A 10 µM solution of EX_31 (pH 7.4) was added to a donor chamber. Samples at multiple
timepoints (up to 90 min) were collected from the donor and receiver chamber for analysis of
the permeability and efflux ratio.
Animals
All animal care and experimental protocols were approved by the ethics review committee
for animal experimentation of Boehringer Ingelheim Pharma GmbH & Co. KG. Animal
studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010). The
pharmacokinetic study was carried out at Boehringer Ingelheim (Biberach, Germany)
according to license 14-009-G and national animal welfare guidelines. The CCl4 study was
conducted at Boehringer Ingelheim (Biberach, Germany) according to license 13-011-G and
national animal welfare guidelines. The CDAA study was conducted at Gubra ApS
(Hørsholm, Denmark) according to license 2013-15-2934-00784 and national welfare
guidelines. Details on animals, experimental procedures, housing and husbandry as well as
animals numbers are disclosed in the following methods sections.Pharmacokinetic profile
of Ex_31 and target engagement in healthy rats
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The ATX inhibitor Ex_31 was dissolved in water containing cyclodextrin (20%; 1 mg in 4
mL) for intravenous administration. For oral administration, Ex_31 was dissolved in water
supplemented with Tween (0.1%) and Natrosol (0.5%; 6 mg in 2.5 mL). These solutions were
administered to fasted male Han Wistar rats (180-200 g) at doses of 1 mol  kg-1
(intravenous) or 10 mol  kg-1 (oral), respectively. Blood samples (150 µL) were taken
sublingually at 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h. Plasma was isolated by
centrifugation and immediately frozen until analysis of LPA levels and compound exposure
as described below.
Quantification of Ex_31
For the quantification of the exposure of Ex_31 in liver the tissue was homogenized in a
mixture of 0.1% formic acid and 30% acetonitrile/methanol (1:1) in a ratio of 1:4. The
homogenate was prepared in a dry iced cooled Precyllis Evolution (Bertin Technologies)
instrument.
5 µL of this homogenate or 5 µL of EDTA plasma was precipitated with 70 µL 0.1% formic
acid and 70% acetonitrile/methanol. Protein precipitate was removed by centrifugation.
Subsequently, a 30 µL aliquot of the supernatant was diluted in 150 µL 0.1% formic acid.
Calibrants from 0.5 nM to 10 µM and quality controls at 10, 100 and 1000 nM were prepared
in EDTA rat plasma. Calibrants, quality controls and samples were supplemented with
internal standard and subjected to LC-MS/MS analysis on an API 4000 (ABSciex, Germany).
The instrument was equipped with a 1200 LC-system (Agilent, Germany), a Kinetex C18
column (50 x 2.1 mm, 2,6 µm, 100 Å Phenomenex), Solvent A (0.1% formic acid) and
Solvent B (1:1 acentonitrile/methanol). A gradient profile was applied, starting at 10%
Solvent B, increasing within 2.6 min to 95% and a decrease to 10% from 3.3 min to 4 min.
Ex_31 was monitored by recording the MRM trace 484.2/143.0 with a declustering potential
of 71 and a collision energy of 57. The lower limit of quantification of Ex_31 was 5 nM.
Carbon tetrachloride-induced hepatic fibrosis in rats
CCl4-induced liver fibrosis is one of the most commonly used animal models to mimic liver
fibrosis in rodents. This model is often used to investigate the role of signaling pathways or
individual proteins in fibrosis development by using knock-out animals or pharmacologic
tools. Male Sprague Dawley rats, 6–7 weeks of age (JANVIER LABS, France), were housed
in pairs in a controlled environment (12 h light/dark cycle). All animals had ad libitum access
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to normal chow (KLIBA 3438; Provimi Kliba AG, Switzerland) and tap water. Animals
received carbon tetrachloride (CCl4; 0.25 mL  kg-1) diluted in olive oil by oral administration
three times a week for 6 weeks, followed by 4 weeks of oral administration of Vehicle or
Ex_31 (15 mg  kg-1) twice daily while maintaining the CCl4 regimen. Ten animals received
olive oil as a control whereas 14 animals per treatment group received CCl4. A higher number
of CCl4 treated animals was used due to the increased likelihood of losing animals under
CCl4 treatment during the course of the study. In total two animals had to be euthanized due
to complication directly after one CCl4 injection. The remaining animals were allocated to the
vehicle and the EX_31 group respectively, resulting in 13 animals in each in these groups.
Animals received the compound (5 mL  kg-1) suspended in Natrosol (0.5% )/Tween 80
(0.01%). Before compound treatment, plasma samples were analysed for collagen IV and
18:1 LPA levels and animals were stratified based on these parameters (Supplemental Table
S4). Plasma was obtained by sublingual bleeding from isofluorane anaesthetised animals. At
the end of the study the animals were sacrificed by final bleeding under pentobarbital
anaesthesia. Livers were weighed and blood and liver samples were used for further analysis.
The CCl4 study was conducted at Boehringer Ingelheim (Biberach, Germany) according to
license 13-011-G and national animal welfare guidelines.
Choline-deficient L-amino acid-defined diet-induced liver injury in rats
As shown in previous studies, rats fed a choline-deficient L-amino acid-defined (CDAA) diet
develop a hepatic phenotype that closely mirrors the human NASH pathology including
steatosis, inflammation and fibrosis. Additionally, animals fed a CDAA diet do not suffer
from massive body weight loss that is observed in animals fed a methionine and choline
deficient diet. Male Wistar rats, 6 weeks of age (JANVIER LABS), were housed in pairs in a
controlled environment (12 h light/dark cycle). After acclimatising, animals were fed either a
CDAA diet containing cholesterol (1 %; E15666-94, ssniff Spezialdiäten GmbH, Germany)
or a choline-supplemented L-amino acid-defined (CSAA) diet (E15668-04, ssniff
Spezialdiäten GmbH) ad libitum for 6 weeks. All animals had ad libitum access to tap water.
Following diet induced liver injury, liver pre-biopsies were obtained with animals maintained
under isoflurane anesthesia as previously described (Clapper et al., 2013; Kristiansen et al.,
2016). Rats were anaesthetised with isofluorane (3–5%) and a midline abdominal incision
was made to expose the left lateral lobe. A tissue biopsy of 50–100 mg was taken and fixed in
4% paraformaldehyde overnight for histological assessment. The remaining cut surfaces were
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electro-coagulated using an ERBE VIO 100C electrosurgical unit (Erbe Elektromedizin,
Germany), the liver returned to the abdominal cavity and both the abdominal wall and the
skin were sutured. The animals received carprofen (5 mg  kg-1; Pfizer, USA) and
enrofloxacin (5 mg  kg-1; Bayer, Germany) both before surgery and on postoperative days 1
and 2 to control postoperative pain and infection. Animals were single-housed after the
surgical procedure. Liver biopsies were used for histological stratification of animals into
groups showing similar collagen 1a1 content (Supplemental Table S5). In each group, twelve
animals received the indicated diet and treatment. From Week 7, animals received oral
administration of Vehicle or Ex_31 (15 mg  kg-1) suspended in 0.5% Natrosol/0.01% Tween
80 twice daily and were maintained on the CDAA diet. Control animals received Vehicle and
were kept on the CSAA diet. After 5 weeks, animals were sacrificed by cardiac puncture
under isofluorane anaesthesia. The liver tissue was excised and weighed and blood and liver
samples taken for further analysis. The CDAA study was conducted at Gubra ApS (Hørsholm,
Denmark) according to license 2013-15-2934-00784 and national welfare guidelines.
Measurement of plasma alanine aminotransferase and collagen IV
Plasma aminotransferase (ALT) activity was measured using 80 µL samples collected into
EDTA tubes using a Cobas Integra 400 or Cobas C-111 (Roche Diagnostics, Germany).
Collagen IV was measured from 50 µL of EDTA plasma using the Exocell Immunoassay
collagen IV M (Exocell, USA) according to the instruction manual. Plasma samples and
collagen IV standards were incubated overnight with anti-collagen IV antibody in a murine
collagen IV pre-coated 96-well plate. After a washing step, goat anti-rabbit IgG horseradish
peroxidase (HRP) conjugate was added to the samples followed by a second incubation for
2 h at 24°C on a shaker set to 450 rpm. Wells were washed again before addition of HRP
substrate (100 L). After 10-12 min reactions were stopped by sulphuric acid addition and
sample absorbance was determined at 450 nm using a SpectraMax microplate reader
(Molecular Devices, USA).
Quantification of plasma and liver lysophosphatidic acid
Lysophosphatidic acid was extracted as described previously (Scherer, Schmitz & Liebisch,
2009). In brief, 35 µL of plasma was transferred to a 96-deep-well plate together with
disodiumhydrogenphosphate buffer (200 µL of 40 mM) containing 30 mM citric acid (pH 4),
followed by addition of 1 µM 17:0 LPA internal standard. To extract the phospholipids,
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500 µL 1-butanol was added to samples, shaken vigorously for 10 min on a monoshake
(Thermo Electron, Germany) and centrifuged at 4°C for 10 min. A sample of the upper
butanolic phase (400 L) was transferred to a 96-deep-well plate and evaporated by an
ultravap (Porvair, UK) with a 60°C heated nitrogen flow (15 psi for 45 min) until dry. The
extract was dissolved in ethanol (100 µL) and LPA levels were determined by mass
spectrometry (MS) as described previously (Bretschneider et al., 2017). An API 6500 mass
spectrometer (AB Sciex, Germany) was equipped with an Agilent 1290 LC system, a CTC
autosampler and an Atlantis 50 x 2.1 mm, 3 µm HILIC LC column (Waters, UK) was used to
determine levels of LPA. The instrument operated in negative mode with a source
temperature of 300°C, cad gas = 50, gas 1 = 60, gas 2 = 60 and voltage of -4500 V.
Declustering potential was set to -150 and collision energy to -28. The following MS
transitions for the LPAs were recorded: 16:0 LPA: 409.2/152.8; 18:0 LPA: 437.3/152.8;
18:1 LPA: 435.3/152.8; 18:2 LPA: 433.2/152.8; 20:4 LPA: 457.2/152.8; and 17:0 LPA:
423.5/152.8.
The bi mobile phase chromatographic system was equipped with Solvent A (0.2% formic
acid and 50 mM ammonium formate in water) and Solvent B (0.2% formic acid in
acetonitrile). Separation of LPA and LPC was achieved with a gradient starting from 95%
Solvent B, which decreasing to 75% over 90 sec and to 50% solvent B over a further 12 sec,
with an increase in the flow rate from 500 to 700 µL·min-1. The column was re-equilibrated
at 108 sec by set back of the solvent B concentration to 95%, which was maintained for
42 sec. Lysophosphatidic acid was eluted at 117 sec and LPC at 126 sec with a peak width of
5 sec. Quality control samples from 0.5 nM to 10 µM were measured to check the linearity of
the LC-MS system and to control the robustness of the system. The lower limit of
quantification was 12.5 nM. The absolute LPA levels were calculated and normalized to
mean of the control group to provide comparable datasets.
Histology assessment of livers in hepatic fibrosis models
For histological analysis of the CCl4 treated animals, the right lobe of the liver was sectioned
and fixed in phosphate-buffered 10% formaldehyde. Each formaldehyde-fixed sample was
embedded in paraffin, cut into 4 µm thick sections and stained with haematoxylin and eosin
(H&E), and Masson`s trichrome according to standard procedures. All slides were scored by
the same pathologist using the NASH activity score (NAS) as described previously (Kleiner
et al., 2005). A semi-quantitative analysis of steatosis, lobular inflammation and
hepatocellular ballooning was assessed using the H&E stained sections. Fibrosis staging was
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performed on the Masson`s trichrome stained liver samples. For quantitative analysis of
collagen positive area and degree of steatosis, histological slides were systematically scanned
with a Zeiss AxioScan.Z1 microscope (Zeiss, Germany). Images were analysed using a script
based on HDevelop ImageAnalysis Toolbox (MVTec, Germany). Image segmentation was
performed using texture and colour information from colour space transformation RGB to
HSI and from colour deconvolution. In the images, liver sections were segmented and the
area covered by liver segmented into mosaic tiles of size 1024*1024 pixels (from 500 to 1200
tiles per slide). For each tissue tile the total area of tissue and the area with collagen rich
tissue was detected and used in calculation of a value describing fibrosis. The median value
of all tiles was reported.
For the histological assessment of samples from the CDAA study, liver pre- and post-biopsies
from the left lateral lobe were fixed in paraformaldehyde (4%) overnight before paraffin
embedding and sectioning (3 µm in depth). Sections were stained with H&E, Sirius Red
(Sigma-Aldrich, Germany) and anti-collagen 1a1 antibody (1:300, Southern Biotech, USA; 2
antibody Bright Vision anti-goat, ImmunoLogic, Netherlands). The stained sections were
used for histological assessment and scoring as described above. In addition, collagen 1a1-
stained slices were analysed with Visiomorph software (Visiopharm) for quantification of
collagen-positive area.
Biochemical quantification of hepatic hydroyproline content
Hepatic collagen content in samples from the CCl4 study was determined using 50–100 mg
liver samples from two different lobes after hydrolysis in hydrochloric acid (6 M) for 16 h at
120°C. Samples were cooled to room temperature and centrifuged at 18,000 g for 10 min.
Standards and samples were transferred to 96-well plates and 50 µL of citrate-acetate buffer
was added. After addition of chloramine T solution (100 µL), plates were incubated for 20
min at room temperature before the addition of of Ehrlich’s reagent (100 l; p–
dimethylaminobenzaldehyde in ethanol:hydrochloric acid). Assay plates were incubated at
65°C for 15 min and then cooled to room temperature. Sample absorbance was measured at
558 nm using a SpectraMax microplate reader (Molecular Devices).
From the CDAA study, 25 mg liver tissue samples were transferred to FastPrep tubes
containing zirconium beads and snap frozen in liquid nitrogen. Hydrochloric acid (20 µL per
g liver tissue, 6 M) was added to each sample tube and homogenised using the FastPrep
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homogeniser (MP Biomedicals, USA) for 1 min, followed by 3 min pause and further
homogenisation (1 min). The samples were incubated overnight at 95°C in a Binder oven
(Binder, Germany). The following day, the samples were cooled to room temperature, mixed
on a vortex and centrifuged (17,900 g for 10 min). Supernatants were transferred to tubes pre-
filled with charcoal (Sigma-Aldrich), mixed on a vortex and centrifuged (17,900 g for 10 min)
and the supernatants transferred to Micronic tubes (In Vitro Technologies, USA).
Hydroxyproline content was determined using a Hydroxyproline Assay Kit (QuickZyme,
Netherlands) according to the manufacturer’s instructions.
Quantification of hepatic alpha smooth muscle actin content
Liver extracts (50 mg) were prepared by suspending in 700 µL of MSD lysis buffer (Meso
Scale Discovery, USA) supplemented with protease inhibitor cocktails (Thermo Fisher
Scientific and Sigma-Aldrich). Samples were homogenised for 30 sec at 6000 g, 4°C, using a
FastPrep homogeniser (MP Biomedicals, USA). Homogenates were centrifuged at 10000 g at
4°C for 10 min. Supernatants were adjusted to a protein concentration of 3 mg  mL-1. An
MSD western-replacement method (Meso Scale Discovery) using specific anti- alpha smooth
muscle actin (SMA) antibody was used to quantify SMA in protein lysates. Samples (25 L)
were added to multi-array 96-well plates (high bind, Meso Scale Discovery) and incubated
for 2 h at room temperature with gentle shaking. Non-specific antibody binding was
prevented by incubation of 150 µL of 3% blocking buffer (0.6 g of MSD Blocker A in 20 mL
bidest) for 1 h at room temperature. For the detection of bound SMA, an anti-S
antibody (1:5000; Sigma-Aldrich) and goat anti-mouse sulfo-TAG antibody (1:167; Meso
Scale Discovery) mixture was prepared in blocking buffer (1.5 mL, 3%) and Tris (4.47 mL,
pH 7.4) wash buffer and 25 µL added per well for 1 h at room temperature. Plates were
washed three times using 200 µL of Tris pH 7.4 wash buffer between all steps. Final
detection reaction was initiated by the addition of 150 µL of MSD read buffer T after which
plates were analysed on a SECTOR S 600 plate reader (Meso Scale Discovery)
Real-time polymerase chain reaction analysis of gene expression in rat livers
Tissue samples were treated prior to the isolation of nucleic acids with a phenol-chloroform
extraction protocol. First, samples were homogenised in Lysing Matrix D Tubes containing
700 µL RLT-buffer (Qiagen) for 3 min at 3000 g, using a FastPrep®-24 homogeniser (MP
Biomedicals). Supernatants were transferred to fresh tubes and 700 µL phenol-chloroform-
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isoamyl alcohol were added. Tubes were mixed and centrifuged at 12000 g for 5 min before
addition of chloroform-isoamyl alcohol (500 µL). Tubes were incubated for 3 min at room
temperature followed by another centrifugation step (12000 g for 5 min). Upper phases were
used for the extraction of RNA. Total RNA was isolated using RNeasy 96 Kit (Qiagen)
according to the manufacturer’s instructions. RNA concentrations were analyzed by
NanoDrop ND-1000 UV-Vis spectrophotometer at 260 nm (Thermo Fisher Scientific). Purity
of RNA was confirmed using 260/280 nm ratios and samples were stored at -80°C before
further processing. For RT-PCR, RNA (50–2000 ng) were reverse-transcribed into cDNA
with a high capacity cDNA archive kit (Thermo Fisher Scientific) in accordance with the
manufacturer’s protocol and produced cDNA was stored at -20°C. For the analysis of gene-
specific mRNA expression, cDNA was amplified by real–time PCR with specific primers and
fluorescent labelled probes (Supplemental Table S8) using a thermal cycler (Eppendorf).
Results were evaluated using SDS software version 2.2 (Thermo Fisher Scientific).
Expression levels were normalised against levels of 18s RNA. For calculation of relative
changes in gene expression, values of individual samples were divided by the mean value of
samples from control animals.
Statistical analysis
All data were analysed using GraphPad Prism 7.00 software. Results are shown as individual
values or as mean ± standard error of mean (SEM) or standard deviation (SD). If not
otherwise stated, ordinary one-way analysis of variance with Tukey’s multiple comparison
tests were used to evaluate statistical significance between control and treatment group data
(*p<0.05). In all experiments where Tukey’s multiple comparison tests were applied, F-test
of the ANOVA demonstrated statistical significance (p < 0.05).
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS
Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the
Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2015, Alexander et al.,
2017).
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Results
Effect of LPA on gene expression in primary human hepatic stellate cells
In primary human HSCs, 18:1 LPA at a concentration of 10 M induced a time dependent
increase in alpha smooth muscle actin gene 2 (ACTA2) expression reaching a maximum of -
4.4-fold at 6 h (Figure 1A). Levels of mRNA for connective tissue growth factor (CTGF) had
increased after 1 h (7.6-fold; Figure 1B). Expression of CTGF remained elevated in LPA
treated cells until 6 h. A transient increase in expression of chemokine ligand 2 (CCL2) and
CXC motif chemokine ligand 1 (CXCL1) was observed, reaching maximal levels after 2 h of
stimulation (18.8-fold and 6.6-fold, respectively; Figures 1C and D). By the 4 h and 6 h time
points, expression levels of CCL2 and CXCL1 were lower than at 2 h.
Effect of LPA on Tumor necrosis factor-related apoptosis-inducing ligand-induced
hepatic stellate cell apoptosis
Treatment of primary human HSCs with 30 ng  mL-1 TRAIL for 24 h induced apoptosis, as
determined by a 7.1-fold increase in Caspase3/7 activity (Figure 2). Lysophosphatidic acid
caused a concentration dependent suppression of TRAIL-induced apoptosis with an estimated
50% inhibitory concentration (IC50) value of 3 M, although at concentrations of up to
30 M, 18:1 LPA had no effect on basal Caspase3/7 activity.
Characterisation of a potent and selective autotaxin inhibitor
The potency of the ATX inhibitor Ex_31 was determined in vitro in a biochemical ATX
assay (IC50 27 nM) and a rat whole blood assay (IC50 10 nM) (Table 1, Supplemental
Figure S2, Bretschneider et al., 2017). The selectivity of Ex_31 was evaluated by screening
the compound at 10 M in a diversity target profile (Eurofin Cerep, France, Supplemental
Table S3). The only target which was inhibited by more than 50% was phosphodiesterase
4D2. The IC50 of Ex_31 on phosphodiesterase 4D2 was determined at 4 µM. Clearance of
Ex_31 after single dose intravenous administration (1 mol  kg-1) was 13 mg  mL-1  kg-1
with a half-life of 294 min. Dose normalised maximal concentration of 248 nM at 24 min
(0.4 h) was achieved after a single administration of Ex_31 (10 mol  kg-1). The in vivo IC50
of Ex_31 was estimated to be 33 nM (data not shown).
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Pharmacokinetic profile of Ex_31 and target engagement in healthy rats
Plasma exposure of Ex_31 reached more than 2 M (~60-fold in vivo IC50) 30 min after a
single oral dose (10 mol  kg-1, Figure 3). At 30 min to 240 min post dosing, a reduction of
90% in maximal plasma LPA compared to baseline was observed. Based on these results,
estimates suggested that administration of 15 mg  kg-1 Ex_31 twice daily would result
in >95% reduction in plasma LPA over a 24 h period.
Hepatic expression of LPA receptor 1 and quantification of plasma and liver LPA levels
in models of carbon tetrachloride- and CDAA diet-induced of liver injury
To determine whether pharmacologic inhibition of ATX prevents fibrosis development we
tested Ex_31 in a 10-week model of CCl4-induced liver injury and in a 14-week model of
CDAA diet-induced liver injury. At the end of the CCl4 study, exposures of Ex_31 were
3525±978 nM in plasma and 17223±2591 nM in livers 15-16 h after last compound
administration. In the CDAA study, exposures of Ex_31 were 360±113 nM in plasma and
3332±767 nM in livers 15-16 h after last compound administration. In CCl4-treated rats,
LPAR1 mRNA expression was increased 25.8-fold compared with control animals (p<0.05,
Figure 4A). Exposure of CCl4-treated rats to Ex_31 did not result in significant changes in
LPAR1 mRNA expression. Lysophosphatidic acid levels were increased in plasma (+36%
compared to control, p<0.05, Figure 4B) and in liver samples (+62% compared with control,
p<0.05, Figure 4C) of CCl4-treated rats. Exposure of CCl4-treated rats to Ex_31 resulted in a
decrease in plasma LPA levels of >95%, whereas levels of LPA in liver homogenates were
unchanged. In CDAA diet-fed rats, LPAR1 mRNA expression was increased 12.2-fold
compared with CSAA diet-fed animals (p<0.05, Figure 4D). Exposure of CDAA diet-fed rats
to Ex_31 did not result in significant changes in LPAR1 mRNA expression.
Lysophosphatidic acid levels in CDAA diet-fed rats were increased in plasma (+39%
compared with control, p<0.05, Figure 4E) and in liver samples (+40% compared with
control, p<0.05, Figure 4F). Exposure of CDAA diet-fed rats to Ex_31 resulted in a decrease
in plasma LPA levels by more than 95%, whereas levels of LPA in liver homogenates were
unchanged.
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Effects of Ex_31 on body weight, liver weight and plasma alanine aminotransferase in
models of carbon tetrachloride- and CDAA diet-induced of liver injury
Before Ex_31 administration and at study termination, body weights of CCl4 treated rats and
control animals were comparable (Table 2). At the end of the study, CCl4 treated rats
displayed signs of liver injury, including elevated plasma ALT levels (365.5 vs. 62.6 U  L-1;
p<0.05) and increased liver weights (25.8 vs. 18.0 g; p<0.05) compared to control animals.
Exposure of CCl4 treated rats to Ex_31 did not result in significant changes in body weight,
liver weight or plasma ALT levels. In the CDAA study, body weights between CDAA and
CSAA diet-fed rats were not significantly different, however, CDAA diet-fed animals had
elevated plasma ALT levels (56.3 vs. 40.5 U  L-1; p<0.05) and increased liver weights
(24.9 vs. 15.4 g; p<0.05) compared to CSAA diet-fed rats. Exposure of CDAA diet-fed rats to
Ex_31 did not result in significant changes in body weight, liver weight or plasma ALT levels.
Effects of Ex_31 on histology in a rat model of carbon tetrachloride-induced liver injury
Rats treated with CCl4 presented signs of liver injury and bridging fibrosis (Figure 5A). In
CCl4-treated rats, NAS scores ranged from 4 to 6 (5.1 ± 0.14; Figure 5B) and fibrosis scores
from 3 to 4 (3.5 ± 0.1, Figure 5C). Quantitative histological assessment based on Masson`s
trichrome staining revealed collagen-positive area of 3.7 ± 0.5% in CCl4 treated rats, as
compared with 0.2 ± 0.0% in control rats (Figure 5D). Exposure of CCl4-treated rats to Ex_31
did not result in significant changes in NAS (5.2 ± 0.2), fibrosis score (3.1 ± 0.3) or area of
collagen-positive staining (3.6 ± 0.9%).
Effects of Ex_31 on histology in a rat model of CDAA diet-induced liver injury
Rats fed a CDAA diet presented signs of NASH and bridging fibrosis (Figure 6A). In CDAA
diet-fed rats, NAS scores ranged from 5 to 7 (5.8 ± 0.3; Figure 6B) and fibrosis scores from 0
to 4 (2.7 ± 0.4; Figure 6C). Quantitative histological assessment based on collagen I staining
revealed collagen-positive area of 15.2 ± 2.6% in CDAA diet-fed rats, as compared with
4.7 ± 0.2% in CSAA diet-fed animals (Figure 6D). Exposure of CDAA diet-fed rats to Ex_31
resulted in NAS of 3 to 6 (4.8 ± 0.3), fibrosis scores of 1 to 4 (3.5 ± 0.3), and area of
collagen-positive staining of 18.6 ± 2.7%.
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Effect of Ex_31 on hepatic hydroxyproline and alpha smooth muscle actin content in
models of liver injury
In CCl-treated rats, hepatic hydroxyproline content was 3.3-fold higher (p<0.05; Figure 7A)
and SMA protein levels were 3.5-fold higher (p<0.05; Figure 7B) than in control animals.
Exposure of CCl4-treated rats to Ex_31 did not result in significant changes in hepatic
hydroxyproline or SMA contents. In CDAA diet-fed rats, hepatic hydroxyproline content
was 5.5-fold higher (p>0.05; Figure 7C) and SMA protein levels were 2.9-fold higher
(p<0.05; Figure 7D) than in CSAA diet-fed animals. Exposure of CDAA diet-fed rats to
Ex_31 did not result in significant changes in hepatic hydroxyproline or SMA contents.
Effect of Ex_31 on expression of inflammation and fibrosis marker genes in models of
liver injury
In livers of CCl4-treated rats, mRNA levels of Ccl2, Cxcl1, intergin alpha M (Itgam),
epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (Emr1),
tumour necrosis alpha (Tnfa) and transforming growth factor beta (Tgfb) were increased 6.5-,
3.1-, 25.0-, 6.0-, 9.2- and 33.0-fold, respectively (p<0.05 for all genes; Figure 8A). Exposure
of CCl4-treated rats to Ex_31 did not result in significant changes in mRNA expression levels
of these genes. In livers of CDAA diet-fed rats, mRNA levels of Ccl2, Cxcl1, Itgam, Emr1,
Tnfa and Tgfb were increased 8.3-, 4.3-, 12.8-, 1.8-, 5.3- and 4.6-fold, respectively (p<0.05
for all genes; Figure 8B). Exposure of CDAA diet-fed rats to Ex_31 did not result in
significant changes in mRNA expression levels of these genes.
Discussion
The aim of the present work was to explore the role of the LPA/ATX axis in the pathogenesis
of NASH-related fibrosis by characterisation of the properties of a selective small molecule
ATX inhibitor in preclinical models of advanced liver fibrosis.
Non-alcoholic steatohepatitis is a progressive liver disease characterised by hepatic fat
accumulation and lobular hepatitis in the absence of viral hepatitis, biliary disease or a history
of alcoholism. Persistent inflammation and oxidative stress can result in hepatocyte damage
and HSC activation, ultimately resulting in fibrogenesis (Maher et al., 2016). Expression of
LPARs in fibroblasts has previously been demonstrated (Stortelers, Kerkhoven & Moolenaar,
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2008) and several effects of LPA on HSCs have been described that could contribute to the
development of fibrosis (Tangkijvanich et al., 2002; Yanase et al., 2000).
Effects of LPA on cytokine, chemokine and fibrosis marker gene expression have been
described in mouse embryonic fibroblasts (Stortelers, Kerkhoven & Moolenaar, 2008). We
have demonstrated expression of LPAR1 and to a lesser extent LPAR6 in culture-activated
primary human HSCs from three different donors (Supplemental Figure S1). To investigate
the effect of LPA in primary human HSCs we have treated cells with 10 µM LPA. While
normal serum LPA concentrations are in the range of 1 µM (Baker et al., 2001), these may be
higher under pathological conditions. Based on previously published in vitro studies, 10 µM
represents a 90% effective concentration (Tangkivanich et al., 2002). We have observed up-
regulation of fibrosis related genes (ACTA2 and CTGF) and chemokine-encoding genes
(CCL2 and CXCL1) in 18:1 LPA-treated primary human HSCs. These results suggest that in
vivo, elevated LPA levels could stimulate HSC activation and chemokine-dependent immune
cell recruitment, both of which are hallmarks of human NASH. Since the elimination of
HSCs by TRAIL-induced apoptosis has been reported to protect rats from CCl4 induced liver
injury (Xu et al., 2016), we investigated the effect of LPA on TRAIL-induced HSC apoptosis
in vitro. We observed the effective suppression of TRAIL-induced HSC apoptosis by 18:1
LPA, which may result in increased HSC survival in pathogenic settings and thereby promote
the development of fibrosis. These in vitro observations prompted us to evaluate the role of
the main LPA producing enzyme ATX in the pathogenesis of liver fibrosis in vivo.
Elevated plasma LPA levels have been observed in different acute and subchronic models of
liver injury (Watanabe et al., 2007). To evaluate the potential role of the ATX/LPA axis in
the pathogenesis of liver fibrosis, we tested a selective small molecule ATX inhibitor, Ex_31,
in a 10-week model of CCl4-induced liver fibrosis and in a more metabolic driven 14-week
model of CDAA diet induced liver injury. The rat was used as the species for proof of
concept studies based on the observation that ATX inhibition resulted in an almost complete
depletion in plasma LPA.
In the present study we used Ex_31 as a selective ATX inhibitor with oral bioavailability,
favourable pharmacokinetic properties and in vivo target inhibition. In the CCl4 study we
observed increased hepatic LPAR1 expression as well as plasma LPA levels, indicating an
up-regulation of the LPA signalling pathway as previously described in several preclinical
models of liver injury (Watanabe et al., 2007). Lysophosphatidic acid levels were elevated
not only in plasma but also in liver tissue of diseased animals. Interestingly, while plasma
LPA was reduced by >95% in animals exposed to Ex_31, LPA levels in liver tissue of these
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animals remained unchanged, indicating an ATX-independent pathway for intracellular LPA
production. Indeed, different enzymes including an acylglycerol kinase that produce
intracellular LPA have been described (Pages et al., 2001; Bektas et al., 2005). To further
address this point, we have measured ATX activity ex-vivo in plasma and homogenates of un-
perfused and perfused rat liver sections. As expected, we observed 18:2 LPA synthesis after
incubation of plasma with 10 µM 18:2 LPC (Supplemental Figure S11). While we also
observed 18:2 LPA synthesis in homogenates of un-perfused livers incubated with 18:2 LPC,
we did not detect any LPA synthesis in homogenates of perfused livers. These results indicate
that in healthy rats liver ATX activity is restricted to the plasma and that intracellular LPA
must be synthesized through ATX-independent pathways. These findings may explain our
observation that liver LPA levels were not reduced in Ex_31 treated rats in spite of high liver
compound exposure.
In the CCl4 induced liver injury study, efficient pharmacologic inhibition of ATX did not
result in the consistent improvement of markers for liver injury, inflammation or fibrosis. The
lack of any signs of anti-inflammatory or anti-fibrotic efficacy of Ex_31, despite excellent
target engagement, prompted us to challenge the relevance of the CCl4 model for the
evaluation of a potential role of the ATX/LPA axis in the pathogenesis of hepatic fibrosis.
Continuous CCl4 challenges could result in the activation of HSCs, which may mask a
potential pathophysiological role for LPA, such as HSC activation or protection of HSCs
from TRAIL-induced apoptosis as shown in our in vitro studies. Indeed, strong induction of
HSC proliferation followed by apoptosis has been observed after single dose administration
of CCl4 in Sprague-Dawley rats (Lee et al., 2003).We also tested Ex_31 in a second, diet-
induced model of liver injury in rats. Multiple NASH-related models have previously been
described in the literature (Ibrahim, Hirsova, Malhi & Gores, 2016). Here, we performed a
14-week study with a modified version of a CDAA diet supplemented with 1% cholesterol.
This model was recently used to characterise the efficacy of the PPAR agonist Elafibranor
(Noel et al., 2015). In that study, supplementation of a classical CDAA diet with 1%
cholesterol markedly increased hepatocyte ballooning and fibrosis development. In line with
these observations, the addition of 1% cholesterol to a 15% high-fat diet has previously been
shown to trigger fibrosis development over the course of 30 weeks of exposure (Savard et al.,
2013). In the present CDAA study, plasma LPA levels were increased moderately after 14
weeks of CDAA diet feeding. However, levels of the ATX substrate LPC were markedly
lower in CDAA diet-fed rats compared with rats receiving CSAA diet (Supplemental Figure
S6). As a consequence, plasma LPA/LPC ratios were significantly higher in CDAA diet-fed
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rats than in CSAA diet-fed animals, indicating increased plasma ATX activity. As observed
in the CCl4 study, LPA levels were not only increased in plasma but also in the livers of
CDAA diet-fed rats. While exposure of CDAA diet-fed animals to Ex_31 resulted in a
decrease in plasma LPA by >95%, no lowering of liver LPA was observed. The CDAA study
confirmed the observation made in the CCl4 model that ATX inhibition by Ex_31 had no
beneficial effects on a majority of markers of liver injury, inflammation and fibrosis. The
finding that liver tissue LPA levels were increased in both models of liver injury, but
unchanged in animals exposed to Ex_31, raises the question of whether or not intracellular
LPA could contribute to the pathogenesis of liver injury and fibrosis. Indeed, intracellular
pro-inflammatory effects of LPA have previously been reported in bronchial epithelial cells
(Kalari et al., 2009). To investigate the potential of intracellular LPA to diffuse outside the
cell and bind to LPA receptors, we investigated passive permeability of different LPA species
in a parallel artificial membrane permeability assay. All tested LPA species showed a very
good permeability at pH 5, 6.5, 7.4 (Supplemental Table S7). Therefore, intracellular LPA
could be released from the cells and subsequently contribute to classical LPA receptor-
dependent signalling.
A different ATX small molecule inhibitor developed by PharmAkea (PAT-505) was recently
tested in two diet-induced models of liver injury in mice (Bain et al., 2017). In the STAMTM
model, administration of PAT-505 (10 mg  kg-1) once per day resulted in a significant
reduction in NAS and fibrotic area. However, it was noted that the observed effects were not
dose dependent and thus inconclusive. In a second CDAA high-fat diet model, PAT-505
significantly reduced fibrosis development but had no effect on steatosis, inflammation or
hepatocyte ballooning. When comparing these results with our studies it is worthwhile
mentioning that the mouse models were characterized by lower degrees of fibrosis
development.
The role of ATX in liver injury has also been investigated with hepatocyte-specific
conditional ATX deletion in a 4-week model of CCl4-induced liver injury in AlbENPP2-/-
mice (Kaffe et al., 2017). In this study, mice deficient in hepatic ATX displayed reduced
markers of fibrosis compared to control mice following CCl4 treatment. In contrast, ATX
deficiency had no significant effect on markers of inflammation. In their study, substantial
transient increase in hepatic ATX mRNA expression and protein peaking at 4 weeks of CCl4
treatment was shown. Elevated plasma ATX activity and plasma LPA levels were reduced to
baseline in CCl4-treated (4 weeks) AlbENPP2-/- mice. Interestingly, plasma ATX and LPA
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levels were unaffected in AlbENPP2-/- control animals not receiving CCl4. These data
suggest that the observed increase in plasma ATX and LPA in CCl4-treated mice is derived
from hepatocytes. However, hepatocytes appear not to contribute to basal plasma ATX and
LPA levels in healthy animals. In conclusion, the observation that liver ATX mRNA and
protein levels returned to baseline at 8 and 12 weeks of CCl4 treatment indicates that
hepatocytes may only produce relevant amounts of ATX during the initiation phase of CCl4-
induced liver injury, but not at later stages. This hypothesis is in alignment with our data,
since in our study we treated the animals with ATX inhibitor in a therapeutic intervention
mode from week 6 to 10 of CCl4 treatment. This may explain lack of liver LPA lowering by
the ATX inhibitor in our study.
Similar effects as described with AlbENPP2-/- mice were observed in CCl4-treated mice
exposed to the ATX inhibitor PF-8380 (30 mg  kg-1, intraperitoneally, twice daily). Plasma
ATX activity and liver LPA levels were reduced by approximately 50% in PF-8380-treated
mice. These results are in contrast to our studies, where almost complete inhibition of plasma
ATX did not result in reduced LPA levels in liver tissue. As discussed above, due to the
transient increase in liver ATX expression in CCl4-treated mice peaking at 4 weeks, the
duration of the CCl4 treatment in the two studies may explain the different outcomes.
Furthermore, we used a different species and the relevance of ATX for the generation of LPA
in the liver may be different in mice and rats.
In summary, we characterised the properties of a selective ATX inhibitor, Ex_31, in two
models of advanced liver fibrosis in rats. In both studies, exposure of animals to Ex_31 did
not result in significant changes in inflammation and fibrosis markers in spite of efficient
target inhibition resulting in >95% reduction in plasma LPA levels. These results are in
contrast to previous publications (Bain et al., 2017; Kaffe et al., 2017), where anti-
inflammatory and anti-fibrotic effects of ATX inhibition or depletion have been shown in
models of liver injury in mice. However, in contrast to the published studies we used rats as
species, and our models were characterized by advanced stages of liver fibrosis up to
cirrhosis. Both differences may account for the observed discrepancies between the studies.
Taken together, our findings question the value of ATX as a new target for the treatment of
advanced stages of NASH-related liver fibrosis.
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Author contributions
Manuel Baader was responsible for the general conception of the studies, in vitro studies with
hepatic stellate cells and wrote the manuscript; Tom Bretschneider was responsible for the in
vitro characterisation of Ex_31, pharmacokinetic studies, LPA and LPC analytics; Andre
Broermann was responsible for the CCl4 study and interaction with the contract research
organisation Gubra for the CDAA study; Joerg F. Rippmann contributed to biochemical
readouts and gene expression analysis of the in vivo studies; Birgit Stierstorfer did the
histology assessments; Christian A. Kuttruff selected the ATX inhibitor and oversaw its
synthesis; Michael Mark revised the design of the studies and the manuscript.
Acknowledgements
We thank the corresponding scientists at Gubra (Denkmark) for excellent interaction during
the phase and execution of the CDAA study.
Conflicts of interest
All authors are full employees of Boehringer Ingelheim Pharma GmbH & Co. KG
References
Alexander SP, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE, et al. (2015). The
Concise Guide to PHARMACOLOGY 2015/16: Enzymes. Br J Pharmacol 172: 6024-6109.
Alexander SP, Christopoulos A, Davenport AP, Kelly E, Marrion NV, Peters JA, et al.
(2017). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G protein-coupled
receptors. Br J Pharmacol 174 Suppl 1: S17-S129.
Bain G, Shannon KE, Huang F, Darlington J, Goulet L, Prodanovich P, et al. (2017).
Selective Inhibition of Autotaxin Is Efficacious in Mouse Models of Liver Fibrosis. J
Pharmacol Exp Ther 360: 1-13.
This article is protected by copyright. All rights reserved.

Baker DL, Desiderio DM, Miller DD, Tolley B, & Tigyi GJ (2001). Direct quantitative
analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray
ionization liquid chromatography-mass spectrometry. Anal Biochem 292: 287-295.
Barbayianni E, Kaffe E, Aidinis V, & Kokotos G (2015). Autotaxin, a secreted
lysophospholipase D, as a promising therapeutic target in chronic inflammation and cancer.
Prog Lipid Res 58: 76-96.
Bretschneider T, Luippold AH, Romig H, Bischoff D, Klinder K, Nicklin P, Rist W (2017).
Ultrafast and Predictive Mass Spectrometry–Based Autotaxin Assays for Label-Free Potency
Screening. SLAS DISCOVERY: Advancing Life Sciences R&D.
Bektas M, Payne SG, Liu H, Goparaju S, Milstien S, & Spiegel S (2005). A novel
acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in
prostate cancer cells. J Cell Biol 169: 801-811.
Castagna D, Budd DC, Macdonald SJ, Jamieson C, & Watson AJ (2016). Development of
Autotaxin Inhibitors: An Overview of the Patent and Primary Literature. J Med Chem 59:
5604-5621.
Chu X, Wei X, Lu S, & He P (2015). Autotaxin-LPA receptor axis in the pathogenesis of
lung diseases. Int J Clin Exp Med 8: 17117-17122.
Clapper JR, Hendricks MD, Gu G, Wittmer C, Dolman CS, Herich J, et al. (2013). Diet-
induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical
disease progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol 305:
G483-495.
Ibrahim SH, Hirsova P, Malhi H, & Gores GJ (2016). Animal Models of Nonalcoholic
Steatohepatitis: Eat, Delete, and Inflame. Dig Dis Sci 61: 1325-1336.
Kaffe E, Katsifa A, Xylourgidis N, Ninou I, Zannikou M, Harokopos V, et al. (2017).
Hepatocyte autotaxin expression promotes liver fibrosis and cancer. Hepatology.
Kalari S, Zhao Y, Spannhake EW, Berdyshev EV, & Natarajan V (2009). Role of
acylglycerol kinase in LPA-induced IL-8 secretion and transactivation of epidermal growth
factor-receptor in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 296:
L328-336.
This article is protected by copyright. All rights reserved.

Kato K, Ikeda H, Miyakawa S, Futakawa S, Nonaka Y, Fujiwara M, et al. (2016). Structural
basis for specific inhibition of Autotaxin by a DNA aptamer. Nat Struct Mol Biol 23: 395-
401.
Khanna D, Denton CP, Jagerschmidt a, Jasson M, Distler O, & Allanore Y (2014).
SAR100842, an Antagonist of Lysophaphatidic Acid Receptor 1, As a Potential Treatment
for Patients with Systemic Sclerosis: Results from a Phase 2a Study. In ACR/ARHP Annual
Meeting. Boston, MA.
Kihara Y, Maceyka M, Spiegel S, & Chun J (2014). Lysophospholipid receptor nomenclature
review: IUPHAR Review 8. Br J Pharmacol 171: 3575-3594.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG, & Group NCRRGW (2010).
Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160:
1577-1579.
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. (2005).
Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
Hepatology 41: 1313-1321.
Kondo M, Ishizawa T, Enooku K, Tokuhara Y, Ohkawa R, Uranbileg B, et al. (2014).
Increased serum autotaxin levels in hepatocellular carcinoma patients were caused by
background liver fibrosis but not by carcinoma. Clin Chim Acta 433: 128-134.
Kristiansen MN, Veidal SS, Rigbolt KT, Tolbol KS, Roth JD, Jelsing J, et al. (2016). Obese
diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy.
World J Hepatol 8: 673-684.
Leblanc R, & Peyruchaud O (2015). New insights into the autotaxin/LPA axis in cancer
development and metastasis. Exp Cell Res 333: 183-189.
Lee JI, Lee KS, Paik YH, Nyun Park Y, Han KH, Chon CY, et al. (2003). Apoptosis of
hepatic stellate cells in carbon tetrachloride induced acute liver injury of the rat: analysis of
isolated hepatic stellate cells. J Hepatol 39: 960-966.
Llona-Minguez S, Ghassemian A, & Helleday T (2015). Lysophosphatidic acid receptor
(LPAR) modulators: The current pharmacological toolbox. Prog Lipid Res 58: 51-75.
This article is protected by copyright. All rights reserved.

Luippold AH, Arnhold T, Jorg W, Kruger B, & Sussmuth RD (2011). Application of a rapid
and integrated analysis system (RIAS) as a high-throughput processing tool for in vitro
ADME samples by liquid chromatography/tandem mass spectrometry. J Biomol Screen 16:
370-377.
Maher JJ (2016). Pathogenesis of NAFLD and NASH. In Alcoholic and Non-Alcoholic Fatty
Liver Disease: Bench to Bedside. eds Chalasani N., & Szabo G. Springer International
Publishing Switzerland, pp 71-101.
Nakagawa H, Ikeda H, Nakamura K, Ohkawa R, Masuzaki R, Tateishi R, et al. (2011).
Autotaxin as a novel serum marker of liver fibrosis. Clin Chim Acta 412: 1201-1206.
Noel B, Rigou G, Degallaix N, Daix V, Cambula L, Roudot A, et al. (2015).
ELAFIBRANOR (GFT505) PREVENTS NASH, HEPATIC FIBROSIS AND
HEPATOCARCINOMA IN A NEW DISEASE MODEL. In The Liver Meeting 2015 -
AASLD. San Francisco, CA.
Pages C, Simon MF, Valet P, & Saulnier-Blache JS (2001). Lysophosphatidic acid synthesis
and release. Prostaglandins Other Lipid Mediat 64: 1-10.
Pasquinelli C (2013). Safety and Efficacy of A Lysophosphatidic Acid Receptor Antagonist
(BMS 986020) in Idiopathic Pulmonary Fibrosis. In PFF Summit. San Diego, CA.
Perrakis A, & Moolenaar WH (2014). Autotaxin: structure-function and signaling. J Lipid
Res 55: 1010-1018.
Pleli T, Martin D, Kronenberger B, Brunner F, Koberle V, Grammatikos G, et al. (2014).
Serum autotaxin is a parameter for the severity of liver cirrhosis and overall survival in
patients with liver cirrhosis--a prospective cohort study. PLoS One 9: e103532.
Savard C, Tartaglione EV, Kuver R, Haigh WG, Farrell GC, Subramanian S, et al. (2013).
Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental
steatohepatitis. Hepatology 57: 81-92.
Scherer M, Schmitz G, & Liebisch G (2009). High-throughput analysis of sphingosine 1-
phosphate, sphinganine 1-phosphate, and lysophosphatidic acid in plasma samples by liquid
chromatography-tandem mass spectrometry. Clin Chem 55: 1218-1222.
This article is protected by copyright. All rights reserved.

Sevastou I, Kaffe E, Mouratis MA, & Aidinis V (2013). Lysoglycerophospholipids in chronic
inflammatory disorders: the PLA(2)/LPC and ATX/LPA axes. Biochim Biophys Acta 1831:
42-60.
Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SP, et al. (2016).
The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative
interactions between 1300 protein targets and 6000 ligands. Nucleic Acids Res 44: D1054-
1068.
Stortelers C, Kerkhoven R, & Moolenaar WH (2008). Multiple actions of lysophosphatidic
acid on fibroblasts revealed by transcriptional profiling. BMC Genomics 9: 387.
Tanaka M, Okudaira S, Kishi Y, Ohkawa R, Iseki S, Ota M, et al. (2006). Autotaxin
stabilizes blood vessels and is required for embryonic vasculature by producing
lysophosphatidic acid. J Biol Chem 281: 25822-25830.
Tangkijvanich P, Melton AC, Chitapanarux T, Han J, & Yee HF (2002). Platelet-derived
growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast
migration through focal adhesion kinase. Exp Cell Res 281: 140-147.
van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, van Rooijen MA, Pradere JP, et al.
(2006). Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation
during development. Mol Cell Biol 26: 5015-5022.
Watanabe N, Ikeda H, Nakamura K, Ohkawa R, Kume Y, Tomiya T, et al. (2007). Plasma
lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in
relation to its severity. Life Sci 81: 1009-1015.
Xu F, Zhou D, Meng X, Wang X, Liu C, Huang C, et al. (2016). Smad2 increases the
apoptosis of activated human hepatic stellate cells induced by TRAIL. Int Immunopharmacol
32: 76-86.
Yanase M, Ikeda H, Matsui A, Maekawa H, Noiri E, Tomiya T, et al. (2000).
Lysophosphatidic acid enhances collagen gel contraction by hepatic stellate cells: association
with rho-kinase. Biochem Biophys Res Commun 277: 72-78.
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Figure 1 Effect of LPA on expression of fibrosis and inflammation marker genes in primary
human HSCs. (A) Alpha smooth muscle actin (ACTA2). (B) Connective tissue growth factor
(CTGF). (C) CC-chemokine ligand 2 (CCL2). (D) C-X-C motif chemokine ligand 1
(CXCL1). Data represent relative mRNA levels normalised versus levels of RNA-polymerase
II and calculated as fold change compared to untreated samples at time zero. Data represent
mean  SD of one representative experiment (n=6 samples per group, each measured in
technical duplicate), *p<0.05.
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Figure 2 Effect of LPA on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-
induced apoptosis in primary human HSCs. Data represent relative fluorescence units (RFU)
measured with a Caspase3/7 activity assay after 24 h incubation. Data represent mean  SD
of one representative experiment (n=7 samples per group, each measured in technical
duplicate).
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Figure 3 Pharmacokinetic profile of Ex_31 and plasma LPA levels after 10 mol  kg-1 single
oral dose in rats. Filled dots represent relative levels of the sum of 16:0, 18:0, 18:1, 18:2 and
20:4 LPA normalized versus controls at time 0. Open rhombuses represent plasma exposures
of Ex_31. Data represent values from three individual animals.
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