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DOI: 10.1039/c6tx00376a
Improved hepatic physiology in hepatic cytochrome P450 reductase null
(HRN™) mice dosed orally with fenclozic acid
James A. Akingbasotea, Alison J. Foster* b, Huw B. Jonesb, Rhiannon Davidb, Nigel J
Gooderhamc, Ian D. Wilsonc, and J. Gerry Kennad
a MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, L69 3GE, United
Kingdom
bDrug Safety and Metabolism, Unit 310 - Darwin Building, Cambridge Science Park, Milton Road,
Cambridge, CB4 0WG.
c Section of Computational and Systems Medicine, Department of Surgery and Cancer
Faculty of Medicine, Imperial College London, South Kensington Campus, London
SW7 2AZ United Kingdom.
d Current affiliation: Drug Safety Consultant, Macclesfield, United Kingdom.
Email addresses: [email protected]; [email protected];
[email protected]; [email protected] ; [email protected];
[email protected]; [email protected]
Corresponding author: * Alison Foster: Drug Safety and Metabolism, Unit 310 - Darwin Building,
Cambridge Science Park, Milton Road, Cambridge, CB4 0WG; E-mail:
[email protected]; tel. : +441625234713
Running Title: HEPATIC EFFECTS OF FENCLOZIC ACID IN HRNTM MICE
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Abstract
Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit no functional
expression of hepatic cytochrome P450 (P450) when compared to wild type (WT) mice, but have
normal hepatic and extrahepatic expression of other biotransformation enzymes. We have assessed
the utility of HRN™ mice for investigation of the role of metabolic bioactivation in liver toxicity
caused by the nonsteroidal anti-inflammatory drug (NSAID) fenclozic acid. In vitro studies revealed
significant NADPH-dependent (i.e. P450-mediated) covalent binding of [14C]-fenclozic acid to liver
microsomes from WT mice and HRN™ mice, whereas no in vitro covalent binding was observed in
the presence of the UDP-glucuronyltransferase cofactor UDPGA. Oral fenclozic acid administration
did not alter the liver histopathology or elevate the plasma liver enzyme activities of WT mice, or
affect their hepatic miRNA contents. Livers from HRN™ mice exhibited abnormal liver
histopathology (enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration,
necrosis, inflammatory cell infiltration) and plasma clinical chemistry (elevated alanine
aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities). Modest apparent
improvements in these abnormalities were observed when HRN™ mice dosed orally with fenclozic
acid for 7 days at 100 mg/kg/day. Previously we observed more marked effects on liver
histopathology and integrity in HRN™ mice dosed orally with the NSAID diclofenac for 7 days at 30
mg/kg/day. We conclude that HRN™ mice are valuable for assessing P450-related hepatic drug
biotransformation, but not for drug toxicity studies due to underlying liver dysfunction. Nonetheless,
HRN™ mice may provide novel insights into the role of inflammation in liver injury, thereby aiding
its treatment.
Key words: Fenclozic acid, hepatotoxicity, intestinal toxicity, biotransformation, covalent binding,
cytochrome P450.
3
Introduction
HRN™ mice lack the gene encoding hepatic NADPH-cytochrome P450 oxidoreductase (CYP P450
reductase; POR; EC 1.6.2.4)1, which catalyses the transfer of electrons from cytochrome P450 (P450)
and other endoplasmic reticulum enzymes which use NADPH as co-factor.2,3 Hence HRN™ mice are
deficient in hepatic P450 activities, whereas the activities of other hepatic drug metabolizing enzymes
and extra-hepatic P450 enzymes are unaffected.1,4 In vivo biotransformation studies have shown that
HRN™ mice can provide valuable insights into the role played by hepatic P450s in xenobiotic
biotransformation. For example, formation of the toxic quinoneimine metabolite was not observed
when HRN™ mice were dosed with acetaminophen (paracetamol).1 This model may also prove
especially useful in circumstances where both oxidative P450-mediated metabolism and conjugation
reactions result in the production of reactive metabolites that might cause toxicity. For example, the
nonsteroidal anti-inflammatory drug (NSAID) diclofenac causes hepatotoxicity in patients and is
metabolised in vivo via both P450-mediated oxidative and UDP-glucuronyltransferase (UGT)-
mediated conjugative routes in wild type mice5, humans and other animal species.6 Currently it is
unclear whether oxidation or conjugation of diclofenac, or a combination of both, is responsible for
the hepatotoxicity caused by the drug in patients.
When administered to HRN™ mice, diclofenac was metabolized overwhelmingly to conjugates
(mainly to taurine and glucuronic acid), with little evidence of oxidative metabolism.7 In a previous
study8 we undertook toxicological studies in wild type (WT) and HRN™ mice dosed orally with
diclofenac to assess whether use of this model might provide novel insights into the possible role
played by conjugation of diclofenac to chemically reactive acyl glucuronides in the liver toxicity
observed in human patients. HRN™ mice treated with dose vehicle alone exhibited enhanced hepatic
lipid accumulation and degenerative liver histopathologies that primarily affected bile ducts and
periportal hepatocytes, plus markedly elevated activities in plasma of various liver-derived enzymes,
which were not present in livers from dose vehicle treated WT mice. Unexpectedly, the severities of
4
the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced
markedly when HRN™ mice were dosed orally with diclofenac for 7 days at 30 mg/kg/day.8
A possible explanation for the positive effect of diclofenac administration to HRN™ mice on liver
pathophysiology is via a reduction/inhibition of hepatic inflammation.9,10 To gain further insight into
whether this is a plausible hypothesis, and determine whether the effects were unique to diclofenac or
were seen with other drugs in this class, in the present studies we have explored tissue histopathology
and plasma clinical chemistry in HRN™ and WT mice dosed orally for seven days with dose vehicle
alone or fenclozic acid (Myalex®). Fenclozic acid is a carboxylic acid NSAID with potent analgesic
and antipyretic activities11 whose clinical development was terminated due to liver injury observed in
clinical trials12 and which also is metabolised to chemically reactive metabolites.13,14 Recently it has
recently been reported that miRNA could be sensitive and informative biomarkers of hepatotoxicity;
for example, paracetamol induced toxicity in mice rapidly resulted in a decline in hepatic miRNAs
such as miR-122 and miR-192.15 This observation prompted us also to investigate whether altered
amounts of hepatic miRNAs were evident when HRN™ and WT mice were treated with fenclozic
acid.
Materials and methods
Chemicals
FluothaneTM, fenclozic acid and [14C]-fenclozic acid (purity >99%, specific activity 60.5 mCi/mmol)
were obtained from AstraZeneca. PierceTM BCA Protein Assay Kit was from Thermo Scientific
(Rockford, IL). Formic acid was from VWR International Ltd (Poole, UK). The EnzyChrom™
Alanine Transaminase Assay Kit was from BioAssay Systems (Hayward, CA). The bile acid reagent
was from Diazyme Laboratories (Dresden, Germany) and other clinical chemistry reagents were from
Roche Diagnostics (Burgess Hill, UK). All other reagents and solvents were from Sigma-Aldrich
5
Company Ltd. (Poole, UK) or Fisher Scientific (Loughborough, UK) and were of HPLC grade or
higher.
In vivo studies
The experimentation, transportation and care of the mice were performed in accordance with
approved project license, the relevant national guidelines (UK Animals and Scientific Procedures Act,
1986) and in compliance with AstraZeneca regulations on the ethical treatment of animals. All
experiments were approved by AstraZeneca’s ethical committee. Statistical analysis was undertaken
to determine the minimum number of animals to give 80% power to detect fold changes of
approximately 2-3 fold in ALT/ALP via pairwise comparison of groups with 5% significance level,
using a t-test approach (2-sided) via ANOVA. The animals were individually housed in standard
conditions with freely available access to drinking water and food. All animals were thoroughly
examined before dosing commenced and at weekly intervals from the start of dosing through to
termination. They were also visually inspected at least once daily after dosing, paying particular
attention up to 3 hours after dosing.
The dose levels and route of administration of fenclozic acid were based on information available in
the literature. 12,16 Male C57/Bl6 WT mice (8-10 weeks old (21-25 g), Charles River, Margate, UK)
were treated daily for 7 days, by oral gavage, with dose vehicle alone (0.1 M sodium phosphate
buffer, pH 7.7; Group 1), or fenclozic acid at doses of 50 or 100 mg/kg/day (in the same vehicle), 10
mL/kg (Groups 2 and 3 respectively; n=5 per group). Male HRN™ mice (4–11 weeks old (20-27 g);
Taconic Farms Inc. Germantown, NY, USA) were treated by daily oral gavage for 7 days with
fenclozic acid at a dose of 100 mg/kg/day (Group 5; n=5), or with equivalent volumes of dose vehicle
alone (Group 4; n=4). The HRN™ mice had been developed using C57BL/6J background strain mice
[1]. Blood samples (50 L) were obtained from the animals by tail bleeding on days -1, 2 and 4 of
the study and plasma ALT activities were determined. At 24 h after the final administration of
fenclozic acid or dose vehicle, animals were euthanized under fluothaneTM anaesthesia. Terminal
blood samples were collected for immediate analysis of plasma alanine aminotransferase (ALT),
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alkaline phosphatase (ALP) and glutamate dehydrogenase (GLDH), which was undertaken using a
Roche Modular P800 Chemistry Analyser (Roche Diagnostics Ltd., Burgess Hill, UK). Additionally,
ALT activities of plasma samples were determined using an EnzyChrom™ Alanine Transaminase
Assay Kit (Bioassays Systems, Hayward, CA).
The liver, intestines, oesophagus and stomach/duodenum were examined for any abnormalities and
weighed and tissue samples were processed to paraffin blocks using standard histologic procedures.
Sections were cut and were stained with haematoxylin and eosin (H&E) for histopathologic
examination using light microscopy. The severities of tissue histopathology findings were scored
qualitatively, using the following scheme: Grade 1 = Minimal / very few / very small; Grade 2 =
Slight / few / small; Grade 3 = Moderate / moderate number / moderate size; Grade 4 = Marked /
many / large; Grade 5 = Massive / extensive number / extensive size.
Statistical analysis of data was performed by paired or unpaired t-testing, using GraphPad Prism
version 4.03 (GraphPad Software, San Diego, CA).
To quantify hepatic miRNAs, total RNA was extracted from liver tissue using the MiRVana PARIS
kit (Life Technologies, Paisley, UK) and was quantified using a NanoPhotometer (Implen GmbH,
Munchen, Germany). The ratios A260/A280 and A260/A230 were used to assess RNA quality. Total
RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Life
Technologies, Paisley, UK). Mature miRNA TaqMan assays (Life Technologies) were used to
generate cDNA, which was amplified using the Taqman 2x Universal PCR master mix, No AmpErase
UNG (Life Technologies), with each reaction performed in triplicate. The qPCR data were analysed
using the ABI 7500 Sequence Detection System (Life Technologies) and the comparative Ct Method
(ΔΔCT Method) [17]. Calibration was based on the expression of snoRNA, as per manufacturer’s
instructions.
Preparation of liver microsomes and determination of microsomal cytochrome c reductase activity
7
The preparation of liver microsomes from both WT and HRN™ mouse livers was undertaken as
described by Akingbasote et al.8 Protein concentrations were determined using the Pierce BCA
Protein Assay Kit®.
In vitro covalent binding (CVB) of [14C]-fenclozic acid to liver microsomes
Determination of CVB for [14C]-fenclozic acid was performed as described by Day et al.18 Liver
microsomes (1 mg/mL final protein concentration) were incubated at 37 °C with 10 M [14C]-
fenclozic acid in 0.1 M phosphate buffer supplemented with 10 mM MgCl2, pH 7.0, in the presence or
absence of NADPH (2 mM) or UDPGA (4 mM). In some experiments, 1-aminobenzotriazole (ABT;
1 mM) was also added to the incubation mixture. The final assay volume was 500 μL. After
incubation for 0 and 60 min, aliquots (200 μL) were mixed with 300 μL of chilled acetone and then a
further 500 μL acetone was added. Precipitated proteins were collected onto filter disks using a
Brandel 96-sample cell harvester (Gaithersburg MD, USA), washed with 80% methanol, then
protein-bound [14C] was quantified by liquid scintillation counting as described by Foster et al. 19 Non-
specific binding (NSB) was determined by incubating microsomes in the absence of fenclozic acid
for 1 hour at 37 °C, then ‘back-adding’ 10 µM [14C]-fenclozic acid immediately prior to processing
the samples. Levels of CVB were expressed as pmol equivalents/mg protein.
Results
Abnormal liver histopathology and plasma clinical chemistry of vehicle treated HRN™ mice
WT mice which were dosed orally for 7 days with dose vehicle alone exhibited no abnormalities in
liver histology, other than minimal extents of non-periportal hepatocyte necrosis with inflammatory
cell infiltration and minimal mixed inflammatory cell aggregates (Figure 1A, Table 1). The
frequencies and severities of these findings were in line with those observed historically in WT
8
control mice. As we have reported previously,8 a variety of histopathological findings were observed
in livers from dose vehicle treated HRN™ mice that were not evident in livers from WT mice.
Elevated numbers and sizes of hepatocellular fat vacuoles, increased extents of bile duct proliferation
and periportal hepatocyte necrosis with accompanying inflammatory cell infiltration were observed in
each of four livers from vehicle-treated HRN™ mice, while a few necrotic hepatocyte foci were also
observed in one liver (Figure 1A and C and Table 1). Furthermore, plasma from mice exhibited
markedly elevated ALT and GLDH activities and less markedly elevated ALP activity, when
compared with plasma from vehicle treated WT mice (Table 2).
Hepatic effects of oral administration of fenclozic acid to HRN™ and WT mice
Oral daily administration of fenclozic acid at doses of 50 or 100 mg/kg/day to WT mice did not result
in any compound-related alterations in liver histopathology (Figure 1C and Table 1), in hepatic
expression of miR-122 or in plasma ALT or GLDH activities (Table 2). However, a dose dependent
reduction in plasma ALP activity was observed in fenclozic acid treated WT mice (Table 2).
When compared with HRN™ mice treated with dose vehicle alone, HRN™ mice dosed orally for 7
days with fenclozic acid at 100 mg/kg/day exhibited less severe bile duct proliferation and periportal
hepatocyte necrosis with accompanying inflammatory cell infiltration (Figure 1, compare panels D
and C; Table 1). The modest apparent reduction in severity of liver histopathology in fenclozic acid
dosed HRN™ mice was consistent with plasma clinical chemistry data, which demonstrated a
statistically significant (p<0.05) reduction in plasma ALP activity and by reductions in plasma ALT
and GLDH activities that did not achieve statistical significance (Table 2). However, the relative
expression of hepatic miR-122 in these animals was unaffected by fenclozic acid treatment (Table 2).
Upper GI histopathology in fenclozic acid treated HRN™ and WT mice
Neither HRN™ nor WT mice dosed orally for 7 days with dose vehicle alone exhibited abnormalities
in upper gastrointestinal (GI) histopathology, apart from minimal or slight oesophageal muscle cell
alterations and minimal glandular epithelial cysts at the stomach limiting ridge in several animals
9
(Table 3). The frequencies and severities of these upper GI findings in these animals were in line
with those observed historically in WT control mice.
No alterations in upper GI histopathology were observed in WT mice dosed with fenclozic acid at 50
mg/kg/day for 7 days. However, three of four WT mice dosed with fenclozic acid at 100 mg/kg/day
exhibited evidence of stomach pyloric erosion and pyloric gastritis was observed in one of these
animals (Table 3). Similar stomach lesions were observed in three of five HRN™ mice dosed with
fenclozic acid at 100 mg/kg/day (Table 3).
In vitro CVB of [14C]-fenclozic acid to liver microsomes from WT and HRN™ mice
CVB of radiolabel to proteins (>170 pmol/mg protein) was observed when [14C]-fenclozic acid was
incubated for 60 min with liver microsomes from WT mice that were supplemented with NADPH
(Table 4). In contrast, CVB to proteins was not observed when equivalent incubations were
undertaken either in the absence of NADPH, or in the presence of UDPGA in place of NADPH.
Similarly, no CVB to proteins was seen when the incubation with NADPH was undertaken for 0 min
or when [14C]-fenclozic acid was “back-added” to microsomes that had been incubated with NADPH
for 60 min. A marked and statistically significant reduction in the amount of NADPH-dependent
CVB to proteins (to <50 pmol/mg protein) was observed when [14C]-fenclozic acid was incubated for
60 min with WT liver microsomes in the presence of the CYP inhibitor ABT. Detectable CVB was
also observed when [14C]-fenclozic acid was incubated for 60 min with liver microsomes from
HRN™ mice in the presence of NADPH. However, the amount of CVB to HRN™ mouse liver
microsomes was approximately 6-fold lower than the amount of CVB to WT liver microsomes.
Furthermore, the CVB to HRN™ mouse liver microsomes was not altered in the presence of ABT. A
detectable amount of CVB was not observed when [14C]-fenclozic acid was incubated for 60 min with
liver microsomes from HRN™ mice in the presence of UDPGA (Table 4).
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Discussion
The clinical development of fenclozic acid was terminated due to liver injury in clinical trials, 12 which
was not observed in any of the animal species used in its preclinical safety testing. In order to gain
insight into the mechanisms responsible for the human-specific hepatotoxicity of this drug, previously
we investigated its potential for bioactivation in vitro and in vivo. The in vitro studies were
undertaken using isolated human hepatocytes13 and also rat, dog and human liver microsomes.14
These revealed relatively high levels of NADPH-dependent CVB to liver proteins, implying that
oxidative bioactivation had occurred. In addition, in vivo metabolite profiling studies identified a
variety of oxidized and glutathione-derived metabolites in urine and bile from fenclozic acid treated
rats20 and WT mice (Pickup et al., in preparation). These findings provided evidence that metabolic
bioactivation of fenclozic acid also occurs in vivo and support our suggestion13,14 this process could be
responsible for the hepatotoxicity caused by the drug in humans in vivo. Such oxidative metabolites
were not seen in urine or faeces obtained from HRN™ mice that had been dosed with fenclozic acid,
where its metabolism was via the carboxylic acid moiety and resulted in the formation of an acyl
glucuronide as well as glycine and taurine conjugates.7 These various metabolic pathways are
summarised in Figure 2.
The NADPH-dependent CVB of radiolabelled fenclozic acid to WT liver microsomal proteins that we
observed in our present investigations are consistent with these earlier studies. However, the small but
statistically significant amount of CVB to proteins in liver microsomes from HRN™ mice, which was
unaffected by addition of the P450 inhibitor ABT, was a novel and unexpected finding. A plausible
explanation is that fenclozic acid is bioactivated in livers from HRN™ mice by a novel mechanism,
which is distinct from the P450 mediated process evident in livers of rats and WT mice and shown in
Figure 2. Additional studies will be required to characterise the responsible enzyme, although a
possible candidate is flavin monoxygenase (FMO).21 Since we observed no evidence of compound
related liver toxicity following in vivo administration of fenclozic acid to either WT or HRN™ mice,
our current investigations were unable to provide insight into the possible relevance of metabolic
bioactivation to its human hepatotoxicity.
11
Similar patterns and severities of adverse stomach pathology were observed when both WT and
HRNTM mice dosed orally with fenclozic acid for 7 days at 100 mg/kg/day, which was not observed in
the vehicle treated animals. The likely explanation for this toxicity is impaired wound healing which
arises as a consequence of cyclooxygenase inhibition, since such toxicity is a common finding in
animals and humans dosed with numerous other NSAIDs.22 However, whereas vehicle-treated
HRNTM mice exhibited no abnormalities in stomach and gastrointestinal pathology when compared
with WT mice, as we have noted previously8 these animals have a variety of underlying abnormalities
in liver pathology and function (resulting in markedly abnormal plasma clinical chemistry).
Therefore whilst HRNTM mice are useful for dissecting out the role of hepatic P450 dependent
processes in drug metabolism, they appear to be less well suited to investigations of mechanisms of
hepatic drug toxicity.
Interestingly, we found no changes in the relative expression of hepatic miR-122 in either mouse
strain, irrespective of treatment. Paracetamol-induced hepatotoxicity has been reported to reduce the
expression of the abundant hepatic enriched miR-122 with a concomitant release of miR-122 into the
plasma.15 This finding prompted the proposal that changes in miRNA expression could be sensitive
and informative biomarkers of drug-induced liver injury. Although the data reported herein fail to
support this hypothesis, there are significant experimental differences between our study and
paracetamol induced toxicity. Specifically, the acute response to paracetamol toxicity and the slower
onset of hepatotoxicity induced by fenclozic acid in humans.12 In addition, the hepatic miR-122 levels
were assessed after 7 days of fenclozic acid administration to mice. It is also pertinent to note that the
hepatic miR-122 levels were unaffected by fenclozic acid treatment of the HRNTM mice, despite the
apparent drug-induced improvement of hepatic histology; again hepatic levels of miR-122 were
assessed after 7 days of treatment. This lack of effect on hepatic miR-122 expression in both mouse
strains may be a reflection of the regulatory function of the miRs and their role in maintaining
homeostasis. In this context, it would be interesting to measure circulating and hepatic miR-122
expression during the first 24 h of treatment of mice with fenclozic acid.
12
The abnormal and degenerative liver histopathology and high plasma activities of liver-derived
enzymes present in vehicle treated HRN™ mice are likely to be a consequence of impaired lipid
metabolism and bile acid homeostasis, which arise due to the absence of hepatic P450 activities in
these animals.1-3 Previously we reported a marked reduction in both degenerative liver
histopathology, and plasma liver-derived enzyme activities (ALT, GLDH and ALP), when HRN™
mice were dosed orally for 7 days with diclofenac.8 Our present results demonstrate more modest
apparent improvements in plasma liver-derived enzyme activities and liver histopathology when
HRN™ mice were dosed orally for 7 days with a second NSAID, fenclozic acid, at a dose of 100
mg/kg/day. Additional experiments are required to clarify whether the apparent effects observed in
HRN™ mice dosed with fenclozic acid can indeed be attributed to compound administration.
Nonetheless, both diclofenac and fenclozic acid have, by design, anti-inflammatory properties and are
potent inhibitors of the enzyme cyclooxygenase, which converts arachidonic acid to prostanoid
inflammatory mediators.23 Inflammatory responses in the liver have been found to mediate hepatic
injury caused by endotoxins and various other xenobiotics.24,25 Hepatic inflammation involves
activation of Kupffer cells, which release biologically active compounds (including cytokines, growth
factors, arachidonic-acid-derived inflammatory mediators and lysosomal enzymes) that cause
hepatocyte necrosis and liver inflammation. Inhibition of cyclooxygenase in Kupffer cells has been
found to impair their activation and it has been proposed that NSAIDs may provide a promising
strategy for mitigation of the liver inflammation, and its resulting liver pathologies, that occurs in
human non-alcoholic fatty liver disease (NAFLD)26,27 and other liver types of liver disease.28
Therefore our current findings are consistent with our previous proposal8 that inflammatory processes
may play important roles in the liver injury evident in HRN™ mice, and that the positive effects of
diclofenac on the liver histopathology and function of these animals could be due to the anti-
inflammatory activity of the drug. It is also conceivable that the liver abnormalities evident in
HRN™ mice might provide a useful model system for exploration of mechanisms of NAFLD and
other clinically important human liver diseases, which could aid the assessment of the potential
therapeutic value of NSAIDs or other novel strategies for treatment of liver inflammation.
13
Acknowledgements
We thank Jonathan Greenall, Elizabeth Johnson and Peter Cotton for their excellent technical
assistance, James Noakes for his directing of the in vivo study and Marie South for her statistical
advice.
Conflicts of interest
The authors declare no conflicts of interest.
Abbreviations
HRNTM, hepatic cytochrome P450 reductase null mice; WT, wild type C57BL6J mice; P450,
cytochrome P450; NADPH, reduced nicotinamide adenine dinucleotide phosphate; UDPGA, uridine
5'-diphospho-glucuronic acid; ALT, alanine aminotransferase; AST, aspartate aminotransferase;
GLDH, glutamate dehydrogenase; ALP, alkaline phosphatase; NAFLD non-alcoholic fatty liver
disease, miR-122, liver specific miRNA 122; snoRNA, small nucleolar RNAs.
14
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18
Table 1. Summary of effects of fenclozic acid administration on liver pathology in wild type and HRN™ mice
Histopathology finding Severity Number of WT mice affected Number of HRN™ mice affected
Vehicle
control
(n=5)
50 mg/kg/day
fenclozic acid
(n=5)
100 mg/kg/day
fenclozic acid
(n=5)
Vehicle control
(n=4)
100 mg/kg/day
fenclozic acid
(n=5)
Hepatocellular fat vacuolation Grade 2 0 0 0 0 0
Grade 3 0 0 0 4 4
Grade 4 0 0 0 0 1
Multifocal hepatocellular
necrosis
Grade 2 0 0 0 1 1
Bile duct proliferation Grade 1 0 0 0 0 2
Grade 2 0 0 0 1 1
Grade 3 0 0 0 3 2
Periportal hepatocyte necrosis/
inflammatory cell infiltration
Grade 1 0 0 0 0 3
Grade 2 0 0 0 4 1
Hepatocyte necrosis with
inflammatory cell infiltration
Grade 1 1 0 0 2 4
Grade 2 0 0 0 0 1
Mixed inflammatory cell
aggregates
Grade 1 2 3 3 0 0
19
Table 2. Plasma ALT, GLDH, ALP and miR-122 in wild type and HRN mice treated with dose
vehicle or fenclozic acid. Values are means ± SD.
Parameter WT mice treated with: HRN™ mice treated with:
Dose vehicle
(n=5)
Fenclozic acid,
100 mg/kg (n=5)
Dose vehicle
(n=4)
Fenclozic acid,
100 mg/kg (n=5)
ALT (IU/L) 37.6 ± 12.6 66.4 ± 25.8 1,764.0 ± 859.7 1,131.6 ± 1056.3
AST (IU/L) 56.0 ± 21.5 71.2 ± 22.3 1,153.0 ± 656.2 965.6 ± 1,022.7
ALP (IU/L) 162.4 ± 35.1 128.0 ± 24.7 444.0 ± 72.7 325.6 ± 31.1 *
GLDH (IU/L) 15.2 ± 9.8 16.8 ± 5.2 521.5 ± 332.08 174.4 ± 152.8
miR-122
(relative expression)
1.0 0.96 1.0 0.86
* p<0.05, compared with vehicle control value.
20
Table 3. Effects of fenclozic acid administration on gastrointestinal histopathology of wild type and HRN™ mice
Organ Histopathology
finding
Severity Number of WT mice affected Number of HRN™ mice
affected
Vehicle
control
(n=5)
50 mg/kg/day
fenclozic acid
(n=5)
100 mg/kg/day
fenclozic acid
(n=5)
Vehicle
control
(n=4)
100 mg/kg/day
fenclozic acid
(n=5)
Oesophagus No abnormalities 0 2 0 4 3 3
Adjacent muscle
degeneration/
necrosis/inflammation
Grade 1 2 0 0 1 0
Grade 2 1 0 1 0 1
Stomach No abnormalities 4 4 1a 4 2
Pyloric ulceration Grade 2 0 0 0 0 1
Pyloric erosion Grade 1 0 0 2 0 0
Grade 2 0 0 1 0 2
Pyloric gastritis Grade 1 0 0 1 0 0
Grade 2 0 0 0 0 2
Glandular epithelial
cyst(s) at limiting ridge
Grade 2 1 1 0 0 0
21
a Tissues from 4 animals were evaluated.
22
Table 4. In vitro CVB of [14C]-fenclozic acid to liver microsomes from wild type mice (WT
microsomes) but not HRN™ mice (HRNTM microsomes). Incubations were undertaken for 0 min (T =
0) or 60 min (T = 60), in the absence (- NADPH) or presence (+ NADPH) of NADPH, in the presence
of NADPH plus ABT (+ NADPH, + ABT), or in the presence of UDPGA (+ UDPGA). Back added =
[14C]-fenclozic acid was added immediately prior to processing the samples. Mean values ± SD from
three independent experiments, each conducted in duplicate, are shown.
pmol equivalents/mg protein
WT microsomes HRNTM microsomes
Mean SD Mean SD
Mic -NADPH, T=0 7.7 2.3 6.0 0.8
Mic -NADPH, T=60 9.7 1.9 9.8 1.7
Mic +NADPH, T=0 19.4 4.6 9.0 2.9
Mic +NADPH, T=60 187.7 * 30.4 32.2 * 7.0
Mic + ABT, T=0 9.7 1.7 10.4 0.0
Mic + ABT, T=60 39.5 # 10.0 29.2 4.2
Mic +UDPGA, T=0 7.1 1.9 8.2 1.9
Mic +UDPGA, T=60 10.4 2.3 9.2 2.8
Back added 33.0 8.5 16.3 4.6
*significantly elevated vs. NADPH background value (+NADPH, T=0); p<0.001#significantly reduced vs. CVB in absence of ABT (+NADPH, T=60); p<0.001
23
Figure 1. Liver histopathology in wild type (A,B) and HRN™ (B,D) mice treated for 7 days with
dose vehicle (A,B) or 100 mg/kg/day fenclozic acid (C,D). Representative photomicrographs from
formalin fixed and H&E stained liver sections at x 20 magnification are shown. The positions of
centrilobular (CL) and periportal (PP) hepatocytes are indicated.
PPCL
PPCL
C. HRN + vehicle
D. HRN + fenclozic acid
CLPP
CLPP
A. WT + vehicle
B. WT + fenclozic acid
24
Figure 2. Metabolism of fenclozic acid. FMO, flavin monooxygenase; CYP, cytochrome P450; UGT,
UDP-glucuronyltransferase; GSH, reduced glutathione.
25
