The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG
mice
C.E. Wilsona, A.P. Dickieb, K. Schreiterc, R. Wehrc, E.M. Wilsond, J. Biald, N. Scheere, I.D. Wilsonf, R.J. Rileyg
aNestlé Skin Health R&D, Les Templiers, Route des Colles BP 87, F-06902 Sophia-Antipolis, FrancebEvotec (UK) Ltd, 114 Innovation Drive, Abingdon, Oxfordshire, OX14 4RZ, UK cEvotec International GmbH, Manfred Eigen Campus, Essener Bogen 7, Hamburg, Germany dYecuris Corporation, PO Box 4645, Tualatin OR 97062, USA eCEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Str. 60-62, 51105 Cologne, GermanyfDept. of Surgery and Cancer, Imperial College, London, UKgEvotec (UK) Ltd, Alderley Park, Nether Alderley, Cheshire, SK10 4TG, UK
Corresponding author:
Dr. Claire E. Wilson: ORCID: 0000-0003-4981-2408
[email protected]; [email protected]
France : 00 33 (0)6 87 95 07 18
ORCID numbers for co-authors:
Dr. Ian D. Wilson ORCID: 0000-0002-8558-7394
Dr. Nico Scheer ORCID: 0000-0002-8847-0415
Abstract
The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric
liver-humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were
investigated in chimeric liver-humanized and murinized FRG mice. Diclofenac reached maximum blood
concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg.h/mL and an effective
half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as
3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h
(n = 3). In C57BL/6J mice mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose
with a mean AUCinf of 2.10 ± 0.49 µg.h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated
only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic
profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and
murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated
metabolites detected. The profiles in humanized mice were different to those of both murinized and wild type
animals e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much
reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study
with previously published data from C57BL/6Jmice and humans, revealed a greater, though not complete, match
between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a
model for assessing the biotransformation of such compounds in humans.
Key words: Liver, humanized mice, metabolism, pharmacokinetics, reactive metabolites.
Introduction
Despite its relatively infrequent occurrence, drug-induced liver injury (DILI) is the leading cause of acute liver
injury in the US (Kola et al. 2004; Waring et al. 2015). Although much of the DILI observed is due to
acetaminophen overdose, non-steroidal anti-inflammatory drugs (NSAIDs), estimated to be used by more than
1% of the US population on a daily basis (Lichtenstein et al. 1995), are also seen as a common cause of
hepatotoxicity. Hepatocellular injury is the most common pattern seen with NSAID hepatotoxicity and
diclofenac (DCF) is the most frequently implicated agent (Schmeltzer et al. 2016). Serious liver injury has been
reported to occur in 6.3 per 100 000 DCF users (de Abajo et al. 2004). DCF was introduced in the UK in 1979
and, despite the potential for DILI, is available without prescription as a generic drug in a number of
formulations.
There are several metabolic pathways which have been implicated in DCF-related DILI in man, chiefly reactive
quinone-imine intermediates generated via CYP450 metabolism (predominantly CYP2C) that can form adducts
with cellular macromolecules unless detoxified via reaction with glutathione (Tang et al . 1999;
Zhou et al. 2009). Alternatively, conjugation of the carboxylic acid moiety of DCF (and its metabolites) to form
acyl glucuronides (DCF-AG) results in a metabolite which can form protein adducts by a variety of mechanisms
(Boelsterli 2003), and it has been suggested that this can also cause toxicity. The underlying mechanisms and
the determinants of susceptibility to DCF-induced DILI remain poorly defined. One proposed mechanism, in
addition to simple damage caused by reaction with cellular macromolecules, is the hapten hypothesis
(Park et al. 1987), a widely accepted theory to explain DILI that suggests that these adverse drug reactions are
mediated by an adaptive immune response (Uetrecht 2003). Autoantibodies have been detected both in patients
who developed DILI after exposure to DCF and those who did not (Aithal et al. 2004). Experimental support for
the hapten hypothesis is incomplete, and an animal model of liver injury and the involvement of antibodies or
sensitized T cells (i.e., an adaptive immune response) has not emerged so far (Shaw et al . 2010). There is also
evidence linking DCF with impaired mitochondrial function or induced mitochondrial permeabilization
(Boelsterli and Lim 2007; Petrescu and Tarba 1997; Bort et al. 1998; Masubuchi et al. 2002;
Gomez-Lechon et al. 2003a, b; Lim et al. 2006). However, it is important to note that mitochondrial dysfunction
can be induced by a number of independent factors such as concurrent xenobiotic exposure, hypoxia, or
inflammation (Shaw et al. 2010). Genetic polymorphisms in pro- or anti-inflammatory cytokines or in anti-
apoptotic proteins could also render individuals more susceptible to DILI involving DCF, where patients who
developed hepatotoxicity were more likely to have polymorphisms in IL-10 or IL-4 expression
(Aithal et al. 2004). Furthermore, genetic polymorphisms in drug metabolizing enzymes (CYP2C8, UGT2B7,
GST), and hepato-canalicular transporters (MRP2, MRP4) have been shown to play a role in the metabolism
and disposition of DCF (Daly et al. 2007; Martinez et al. 2007; Reid et al. 2003). More recent results support the
fact that several hepatic and renal transporters may play an important role in the distribution and elimination of
DCF-AG (Zhang et al. 2016) and multifactorial pathways by which DCF-AG can act as a direct contributor to
toxicity following DCF administration have been proposed (Scialis and Manautou 2016).
DILI remains a major clinical problem and poses considerable challenges and associated costs for drug
development. Although the incidence for a particular drug may be very low, the outcome of DILI can be serious.
Unfortunately, prediction has remained poor (both for patients at risk and for new chemical entities) (Boelsterli
and Lim 2007). Chimeric liver humanized mouse models, where human hepatocytes have been reported to
replace >90 % of the murine hepatocytes, have been shown to have value for various applications in drug
metabolism and toxicity (Kamimura et al. 2010; Strom et al. 2010; Scheer and Wilson 2016). Our hypothesis for
undertaking the studies described here is that the metabolism of DCF by chimeric humanized and murinized
FRG mice would show a similar pattern to that observed in vivo in human and mouse respectively and could
therefore be of use as a model to study DCF-related human DILI. In order to assess this hypothesis the
metabolite profiles of DCF in chimeric humanized and murinized FRG mice were compared with those
previously described for normal C57BL/6J mice (Sarda et al. 2012, 2014) and the published data for humans
(Willis et al. 1976; Riess et al. 1978; Stierlin et al. 1979a, b; Faigle et al. 1988; Degen et al. 1988; Poon et al.
2001; Zhang et al. 2016).
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2. Materials and methods
2.1. Chemicals
Diclofenac was purchased from Selleck Chemicals LLC (supplied by Absource Diagnostics GmbH, Munich,
Germany) and used as supplied. Diclofenac acyl glucuronide standard was synthesized in house at AstraZeneca
(Alderley Park, UK). 4’-hydroxydiclofenac and 5-hydroxydiclofenac were purchased from Santa Cruz
Biotechnology Inc. (Dallas, Texas, USA) and 2-(2-nitro-4-trifluoro-methylbenzoyl)1,3-cyclohexedione
(NTBC) was supplied by Yecuris (Tualatin, OR, USA). Tolbutamide, ammonium acetate and formic acid were
purchased from Sigma-Aldrich (Dorset, UK) and leucine enkephalin was supplied by Waters Ltd (Elstree, UK).
Analytical grade acetonitrile containing 0.1% formic acid, along with unmodified acetonitrile and methanol,
were supplied by Fisher Scientific UK Ltd (Loughborough, UK).
2.2. Animal studies
All animal procedures were performed in accordance with Annex III of the Directive 2010/63/EU applying to
national specific regulations as specified in the German law on animal protection. The PK of DCF in C57BL/6J
mice was investigated in 3 male mice, 8 weeks of age supplied by Charles River Laboratories (Sulzfeld,
Germany). The PK and the routes, rate of excretion and metabolic fate of DCF were investigated in 7 male
chimeric humanized mice (Hu-FRGTM) and 7 male chimeric murinized mice (Mu-FRGTM) (30 g), (FRG
KO/C57BL/6J) (Yecuris (Tualatin, OR, USA)). Following receipt, the mice were group housed in cages of up to
3 and maintained under a 12 h light/dark cycle with free access to food and water, and conditions where
temperature and humidity were controlled. The Hu-FRGTM and Mu-FRGTM animals were initially maintained on
NTBC for 7 days, then removed from NTBC for 4 weeks prior to the first dose, according to the Yecuris
protocol. Before commencing the study a blood sample (25 µL) was taken via the tail vein from each of the
humanized mice in order to assess the human serum albumin (HSA) concentrations, to ensure that the
humanization of the liver was at least ca. 90% according to the Yecuris protocol. Animals were randomized
according to body weight and extent of humanization and then allocated to dosing groups. Groups of two Hu-
FRGTM and Mu-FRGTM mice received dose vehicle (water) whilst each of five Hu-FRGTM and Mu-FRGTM and
three C57BL/6J mice were administered DCF, at a nominal dose of 10 mg/kg, as a solution in water by oral
gavage. Three animals from the Hu-FRGTM, Mu-FRGTM and C57BL/6J groups were taken for the determination
of the PK of DCF. Whole blood (20 µL) was collected pre-dose, and 0.25, 0.5, 1, 2, 4, 6 and 8 h post-dose from
the tail vein into Minivette POCT K-EDTA coated capillaries and then transferred to 96-well plates, pre-
prepared with 20 µL purified water containing 0.2% v/v phosphoric acid (to prevent the
hydrolysis/rearrangement of acyl glucuronides), as soon as possible after collection. Gall bladders were taken
from this PK group upon sacrifice at 8 h post-dose. The remaining two animals from the Hu-FRGTM and Mu-
FRGTM groups were used to investigate the metabolite profile of DCF. The animals were placed individually in
metabolic cages. Urine and faeces for metabolite profiling from animals dosed with DCF were collected, over
dry ice to ensure sample stability, over 0-8 h and 8-24 h time periods. Urine and faeces from animals dosed with
vehicle were collected over dry ice over a 24 h period, and used as controls for metabolite identification.
Samples were frozen as soon as possible after collection on dry ice and stored frozen at -80°C until analysis.
After the final sampling time point (8 h post-dose for the PK group, 24 h post-dose for the metabolite profiling
group) the animals were anaesthetized by isoflurane inhalation and sacrificed by exsanguination. One aliquot, of
up to 500 µL, of Li-Heparin-plasma was obtained from the terminal bleed (in addition to the microsampling
probe). The gall bladder was removed and stored at -80°C until analysis.
2.3. Determination of humanization by ELISA
The level of humanization of each Hu-FRGTM mouse was estimated by measuring human albumin in mouse
plasma using ELISA (Serum Albumin Human, Abcam # ab179887) according to the manufacturer’s protocol.
The same assay was performed on terminal plasma samples to assess continuance of liver humanization during
the study. The results are available in the supplementary data (Table S1).
2.4. Quantitative analysis of diclofenac in blood
2.4.1. Sample preparation
Aliquots of diluted blood (40 µL) and diluted control blood spiked with DCF to provide calibration (30, 100,
300, 1000, 3000, 10,000 ng/mL) and QC samples (40, 400, 4000 ng/mL), were extracted by the addition of 5
volumes (v/v) of cold acidified acetonitrile (ACN) containing 200 nM tolbutamide as an internal analysis
standard, mixed vigorously and centrifuged (4566g, 20 min) and diluted 1:2 (v/v) with water.
2.4.2. Sample analysis
Analysis of DCF in blood was performed by UHPLC-MS/MS using reversed-phase chromatography with a
rapid gradient (1.3 min) on a BEH C18 column (Waters Ltd, Elstree, UK). Mass spectrometric analyses were
conducted on an API 6500 triple quadrupole instrument (AB Sciex UK Ltd, Warrington, UK) operating in
negative ion electrospray ionisation (ESI) and multiple reaction monitoring modes (optimized transition for
DCF was 294 > 250, with declustering potential DP -8 V, entrance potential EP -10 V, collision energy CE -15
V, and collision exit potential CXP -20 V). Non-optimized transitions corresponding to expected metabolites of
DCF were also analyzed simultaneously. The instrument was controlled, and data acquired and processed by
AnalystTM v.1.6.2 (AB Sciex UK Ltd, Warrington, UK). Instrument performance (chromatography and response
of standards) was assessed before and after sample batch injection to ensure system suitability.
2.4.3. Blood pharmacokinetics
Phoenix WinNonlin 6.4 (Pharsight, Mountain View, CA) was used to generate PK parameter estimates using
non-compartmental analysis. Peak (observed) blood concentrations (Cmax) and AUCinf, as determined by the
linear trapezoidal rule were determined per animal and presented as the mean (n = 3 for C57BL/6J mice, n = 3
for Mu-FRGTM mice). For Hu-FRGTM mice, one mouse was terminated 4 h after dosing, so AUCinf and t1/2 are
reported as the average of n = 2.
2.5. Metabolite profiling and identification
2.5.1. Sample preparation
In addition to PK analysis, metabolite profiles were determined using aliquots of diluted blood (40 µL) obtained
from animals pre-dose and 1, 2 and 4 h post-dose. Samples were extracted by the addition of 4 volumes (v/v) of
ACN, with vigorous mixing, and centrifugation (4566g, 20 min) followed by dilution with 1:1 (v/v) with water.
Urine samples obtained for metabolite profiling were pooled by dose group according to weight of urine
collected, for each time range (0-8 h and 8-24 h for dosed animals, 0-24 h for vehicle animals). Pooled urine
samples were centrifuged (20,800g, 5 min) to remove particulates. Gall bladders, removed at 8 h and 24 h, from
dosed animals were extracted with 8 volumes (w/v) of ACN, mixed vigorously, and sonicated for 30 min. The
supernatants were pooled by dose group according to weight of gall bladder, centrifuged (20,800g, 5 min) to
remove particulates and diluted 1:1 (v/v) with water.
Faeces samples were extracted twice with 3 volumes (w/v) of MeOH:H2O 1:1 (v/v) and then with 3 volumes
(w/v) of MeOH (with centrifugation (4566g, 20 min) after each extraction and removal of the supernatant).
Aliquots of the combined supernatants from each sample (0-8 h and 8-24 h for dosed animals, 0-24 h for vehicle
only mice) were pooled by dose group according to the weight of faeces collected and then evaporated from ca.
1 mL to ca. 200 µL under a stream of dry nitrogen at ambient temperature.
2.5.2. Sample analysis
Metabolite profiles and identities were obtained using a reversed-phase gradient HPLC-QTOF-MS/MS method
that had been developed previously to resolve DCF and its murine metabolites (Sarda et al. 2012). Briefly, 50
µL aliquots of samples were separated on a Hypersil Gold C18 column (Fisher Scientific UK Ltd,
Loughborough, UK) with a SecurityGuard C18, 3 µm precolumn filter (Phenomenex Inc., Macclesfield, UK)
and eluted over 60 min. The post-column eluent was monitored by both a photodiode array detector (Waters
Ltd, Elstree, UK) (210-400 nm at 20 spectra/s) and a Xevo G2 Q-Tof mass spectrometer (Waters Ltd,
Wilmslow, UK) operated in positive ion ESI mode. The capillary voltage was set to +500 V, sampling cone to
25 V and extraction cone to 4 V. The source temperature was set to 150 °C, desolvation temperature to 500 °C,
the cone gas flow was set to 50 L/h, and the desolvation gas flow to 1000 L/h. Mass spectrometric data were
collected in resolution mode, in centroid data format, with a scan time of 1 s and a scan range of 50-1200 Th at a
nominal resolution of 30,000. Full scan and product ion mass spectra were acquired simultaneously by HPLC-
QTOF-MSE. Collision energy was applied over a ramp of 20-40 eV for each product ion scan. The instrument
was controlled and data acquired by MassLynxTM v.4.1 (Waters Ltd, Wilmslow, UK). Full scan and product ion
mass spectra were interrogated by extracting chromatograms of potential metabolites using MassLynxTM v.4.1
from the raw data. Representative extracted ion spectra of DCF and its metabolites are available in the
supplementary data (Fig. S1). Comparison was also made with samples from the appropriate control group (or
taken pre-dose) to minimize the potential for false positives from endogenous compounds. The mass
spectrometer was calibrated with sodium formate (5 mM) in positive ion mode, and further aligned using an
internal lock mass of 2 ng/mL leucine-enkephalin ([M+H]+ 556.2771 Th) infused at 10 µL/min and scanned for
1 s every 57 s. Instrument performance (chromatography, response and mass accuracy of standards) was
assessed before and after sample batch injection to ensure data quality. The measured mass accuracy for
standards was less than 5 ppm.
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3. Results
3.1. Clinical signs and degree of humanization
Plasma HSA concentrations were measured at >4 mg/mL in the humanized mice, consistent with the animals
being >90% humanized on days 24 and 29 after removal of NTBC diet. One animal in the Hu-FRGTM mouse
group was terminated 4h after DCF dosing. There were no other clinical observations with all animals behaving
normally following oral administration of either vehicle or DCF at 10 mg/kg..
3.2. Pharmacokinetics of diclofenac
The blood concentration versus time profiles for DCF in the Hu-FRGTM, Mu-FRGTM and C57BL/6J mice are
shown in Fig. 1. After administration of the single oral dose (10 mg/kg) of DCF to Hu-FRGTM mice the drug
was rapidly absorbed, with mean peak blood concentrations of 2.43 ± 0.92 µg/mL being reached at
approximately 0.25 h post-dose. Good, but variable, exposure was achieved with the mean AUCinf determined as
3.67 µg.h/mL (n = 2) and a mean effective half-life of 0.86 h (n = 2): profiles were indicative of enterohepatic
recycling. Similarly following oral dosing of DCF to Mu-FRGTM mice rapid absorption was also seen, with
mean peak blood concentrations of 3.86 ± 2.31 µg/mL being reached at approximately 0.25 h post-dose. Similar
exposure to that seen for the Hu-FRGTM mice was achieved with the mean AUCinf for DCF determined as 4.94 ±
2.93 µg.h/mL and a mean half-life of 0.52 ± 0.03 h (n = 3). Evidence of enterohepatic recycling was observed in
only one Mu-FRGTM animal.
Following oral dosing of DCF to wild type C57BL/6J mice (n = 3) mean peak observed blood concentrations of
2.31 ± 0.53 µg/mL were reached at approximately 0.25 h post-dose. Compared to the Mu-FRG TM mice,
exposure of the C57BL/6J mice was less than 2-fold, with the mean AUC inf determined as 2.10 ± 0.49 µg.h/mL
and a mean half-life of 0.51 ± 0.49 h (n = 3). No evidence of enterohepatic recycling was observed. By way of
comparison to healthy male (n = 6) and female human volunteers (n = 7), when DCF was administered as a
single 50 mg dose, peak plasma concentrations were achieved at 2.5 ± 1.1 h and 1.25 ± 0.58 h post-dose in
female and male subjects, respectively (Willis et al. 1976; Zhang et al. 2016). An apparent mean terminal half-
life for DCF of 1.8 ± 2.1 h was observed in female subjects (Willis et al. 1976) but no data were presented for
male subjects. The PK properties of DCF in Hu-FRGTM, Mu-FRGTM, wild type C57BL/6J mice, and in healthy
male and female subjects are compared in Table 1. Enterohepatic recycling was not observable in the human PK
profiles (data not shown).
3.3. Diclofenac in blood
Only trace quantities of drug-related material were detected in the blood of animals at 24 h post-dose. Thus,
DCF itself and the DCF-AG conjugate (M44) were detected in the blood of both Hu-FRGTM and Mu-FRGTM
mice whilst the taurine conjugate (M48) was only observed in the blood of Mu-FRGTM mice.
3.4. Diclofenac and metabolites in urine
The LC-MS profiles observed for the 0-8 h (data not shown) and 8-24 h urine samples (Fig. 2a) were similar for
Hu-FRGTM mice. The same observation was made for the Mu-FRGTM mice (Fig. 2b). The chromatographic and
mass spectrometric properties of these metabolites are provided in Table 2. A full summary table of HPLC and
mass spectrometric data obtained for DCF and its metabolites in both Hu-FRG™ and Mu-FRG™ mouse urine,
faeces, bile and blood are provided in the supplementary data (Table S2). In the absence of authentic standards it
was not possible to quantify the amounts of each metabolite produced and, as a result only a qualitative
comparison between Hu-FRGTM and Mu-FRGTM mice could be reported.
Extensive metabolism was observed in Hu-FRGTM mice (Fig. 2a), dominated by the signal for the DCF-AG
conjugate (M44), and hydroxy-glucuronide (M35) accompanied by smaller amounts of minor transacylated
glucuronides (M47). Unchanged DCF was also detected (possibly in part derived from hydrolysis of DCF-AG
conjugates) whilst the LC-MS profiles also showed a large number of less intense signals corresponding to a
range of hydroxylated, lactamized and glucuronidated metabolites with small amounts of taurine conjugates,
hydroxymethyl metabolites (M7 and M11) and a riboside (M18) also present.
Extensive metabolism also occurred in Mu-FRGTM mice (Fig. 2b), dominated by the signal for the DCF-AG
conjugate (M44), accompanied by large amounts of direct taurine conjugates (M48). Unchanged DCF was also
detected (again potentially in part derived from hydrolysis of acyl glucuronide conjugates) whilst the LC-MS
profiles also showed, as seen with the Hu-FRGTM mice a range of less intense signals for various hydroxylated
metabolites. For C57BL/6J mice (Sarda et al. 2012), the relative signals for the hydroxyglucuronide (M3) and
taurine (M48) metabolites appeared more intense compared to those seen for Mu-FRGTM mice in the present
study (Table 4). A hydroxy-ribose conjugate (M18) was observed in the urine of Hu-FRGTM, Mu-FRGTM and
C57BL/6J mice (Tables 2 and 4). This metabolite corresponds to the putative hydroxy-riboside (denoted M15)
observed in C57BL/6J mice following a single oral dose of [14C]-DCF at 10 mg/kg previously reported as a
novel conjugation by Sarda et al. (2012) following analysis of urine and faeces. In the present study, M18 was
only found in urine and not faeces or bile.
Also detected in the urine of Hu-FRGTM mice were minor metabolites assigned as cysteine conjugates of
hydroxy-deschloro intermediates (M19 and M21) which were not observed for either the Mu-FRGTM mice, or in
the previous study in C57BL/6J animals. In the urine of Mu-FRGTM mice a deschloro-cysteinyl metabolite
(M26) was observed, whilst in samples from both Mu-FRGTM and Hu-FRGTM mice a hydroxyl-cysteinyl
metabolite (M29) was detected. Glutathione-derived metabolites of this type were not reported by
Sarda et al. (2012) in the urine of C57BL/6J mice.
3.5 Metabolite Profiles of Diclofenac in Bile
The LC-MS profiles observed for the 8 h bile samples from both Hu-FRGTM and Mu-FRGTM mice (Fig. 3)
showed the presence of a number of hydroxylated and conjugated metabolites of DCF. The biliary metabolite
profile for the Hu-FRGTM mice was dominated by the ether glucuronide (M3, M5, M8) and hydroxyglucuronide
(M35) conjugates, dihydroxy-DCF (M15) and an unassigned conjugate (M42), with the cysteine conjugate of a
hydroxy-deschloro intermediate (M19) also present. Other metabolites included the lactamized indolinone forms
of the hydroxyglucuronide conjugates (M27/28), hydroxyglucuronides (M32/33) and hydroxy-DCF (M34).
Unchanged DCF was also detected (possibly in part derived from hydrolysis of the DCF-AG).
The biliary profile of the Mu-FRGTM mice was dominated by hydroxy-glucuronide conjugates (M3, M8), the
DCF-AG (M44) and taurine conjugates (M48). Other metabolites detected in bile included the hydroxy-
mercapturate (M12), hydroxyglucuronide indolinone (M27/28), hydroxy-DCF (M34) and hydroxyglucuronide
(M35). Unchanged DCF was also detected (again, in part possibly resulting from the hydrolysis of the DCF-
AG). In the bile samples obtained at 24 h post-dose, from both types of chimeric mice (metabolite profiles not
shown), only the ether glucuronide conjugates (M3, M8) were detected and these were present only in trace
quantities. Metabolite information is summarized in Table 2.
3.6 Metabolite Profiles of Diclofenac in Faecal Extracts
The LC-MS profiles observed for the faecal extract (8-24 h) from both male Hu-FRGTM and Mu-FRGTM mice
(Fig. 4) showed small amounts of DCF present. In addition, extracts from the faeces of Hu-FRGTM mice
contained hydroxy-DCF (M34) and an unassigned conjugate (M42) which were not present in Mu-FRGTM mice.
Additionally, Mu-FRGTM mouse faecal extracts contained taurine (M48), hydroxy-taurine (M40, M41) and
hydroxy-glucuronide (M24/25) metabolites which were absent from the faecal extracts of the Hu-FRGTM mice.
4. Discussion
The present studies reveal the complexity of the metabolic fate of DCF in both Hu-FRGTM and Mu-FRGTM mice,
involving a broad range of oxidative functionalization reactions and glucuronide, taurine and glutathione
conjugations as summarized in Tables 2 and 3, and depicted in Figs. 2-6. Some of these metabolites have
already been reported as common to both humans and C57BL/6J mice and, as expected, the excreta of both the
chimeric Hu-FRGTM and Mu-FRGTM mice shared a number of metabolites (to facilitate comparison these
metabolite profiles are summarized in a “heat”, or “met”, map” in Table 4). In all three types of mouse model
(wild type, Hu-FRGTM and Mu-FRGTM) and humans, the “universally” detected metabolites included hydroxy
(M34, M36, M37), dihydroxy (M15), hydroxy-glucuronides (M3, M8, M35), AG (M44) and transacylated
glucuronides of DCF (e.g., M47). However, it is also clear that the Mu-FRGTM mice, which had been
repopulated with hepatocytes derived from C57BL/6J mice, whilst producing many of the same metabolites,
were not (based on the results of Sarda et al. 2012, 2014) equivalent to the C57BL/6J animals but also produced
some metabolites seen in the profiles of the Hu-FRGTM mice. Similar observations were made when comparing
the metabolism of lumiracoxib in Hu-FRGTM, Mu-FRGTM, C57BL/6J mice and humans (Dickie et al. 2017).
Such a result may reflect either “strain” differences, gene expression changes in Mu-FRGTM mice compared to
wild type animals, or perhaps extrahepatic “murine” metabolism in e.g., the gut or kidney (although previous
studies in hepatic reductase null (HRN) mice suggest that hepatic metabolism predominates in the mouse
(Pickup et al. 2012)).
Major metabolites in both Mu-FRGTM and C57BL/6J mice were the taurine conjugates (M40, M41 and M48),
which were also detected in much less amounts in the Hu-FRGTM mice (Table 4). In comparing taurine
conjugation between Hu-FRGTM and Mu-FRGTM mice the mean ratio was found to be above 57:1 in favour of
the murinized mice for the direct taurine conjugate, whilst for the hydroxy taurine metabolites the ratio was
25-68:1 in favour of the murinized mice (Table 3). Given the propensity for taurine conjugation in the mouse the
detection of taurine conjugates following administration of DCF to Hu-FRGTM mice may be due to the residual
murine hepatocytes in the liver of these chimeric animals. This apparent “mouse-specific” production of taurine
conjugates from carboxylic acid-containing drugs such as DCF and lumiracoxib (and their metabolites)
(Dickie et al. 2017) may therefore represent a “biomarker” of the residual mouse-specific xenobiotic metabolism
due to the remaining mouse hepatocytes present in the liver of the Hu-FRGTM mice.
In terms of pharmacokinetics, the exposure of DCF in C57BL/6J, Mu-FRGTM and Hu-FRGTM mice was similar.
There was however, evidence of enterohepatic recycling in the PK profiles for DCF in Hu-FRGTM mice (Fig. 1a)
but not in the majority of the Mu-FRGTM mice (Fig. 1b) or C57BL/6J mice (Fig. 1c). Studies in humans
(e.g., Schneider et al. (1990)) described the biliary excretion of small amounts (1 to 9%) of unchanged DCF.
In the present study there was strong evidence for the formation of transacylated products (M38, M43, M45)
from the 1--O-acyl glucuronide of DCF (M44) and this is consistent with previous work with C57BL/6J mice
(Sarda et al. 2012, 2014). The detection of DCF-AG and related compounds may be significant in terms of the
generation of reactive metabolites, given the ability of these acyl glucuronides to react with proteins (Kretz-
Rommel and Boelsterli 1993; Kretz-Rommel and Boelsterli 1994; Hargus et al. 1994; Le et al. 1997; Boelsterli
2003). Indeed, a recent investigation (Hammond et al. 2014) has demonstrated the presence of a complex
pattern of adducts to HSA in samples derived from patients, with various combinations of N-acylations and AG
glycations characterized. The production of the DCF-AG is also significant for intestinal toxicity, as opposed to
DILI, as hydrolysis back to the aglycone, catalysed by bacterial glucuronidases produced by the gut microbiota,
has been shown to cause DCF-induced enteropathy in mice (LoGuidice et al 2012).
The metabolite profiles obtained from the Mu-FRGTM and Hu-FRGTM mice also revealed the presence of
cysteinyl and N-acetylcysteinyl (mercapturate) metabolites, presumably the result of the further metabolism of
glutathione conjugates, often associated with reactive metabolite-related toxicity. A number of these appear to
have resulted from the metabolic dechlorination of DCF, with the pattern seemingly dependent on the nature of
the hepatocytes used to repopulate the livers. Specifically, the hydroxy-deschloro cysteinyl conjugates (M19 and
M21) were only observed in Hu-FRGTM mice whilst the deschloro-cysteinyl conjugate (M26) was only detected
in Mu-FRGTM mice (but not C57BL/6J mice (Sarda et al. 2012, 2014)). The hydroxy-mercapturate conjugate
(M12) was observed in urine and bile of both Hu-FRGTM and Mu-FRGTM mice (but not C57BL/6J mice
(Sarda et al. 2012, 2014)) and has previously been detected as a result of in vitro studies using human liver
microsomes (Kumar et al. 2002). Similarly, the hydroxy-cysteine metabolite (M29) was observed in the urine of
both Hu-FRGTM and Mu-FRGTM mice. The formation of these cysteinyl and mercapturate conjugates provides
evidence for potential toxicity and were observed at different intensities in the present study in both Hu-FRGTM
and Mu-FRGTM mice. The presence of mercapturate and cysteine conjugates (M12, M26 and M29) in Mu-
FRGTM mice was surprising because although such metabolites have been observed in the urine of rats and
humans treated with DCF (Poon et al. 2001), they were not detected in studies on C57BL/6J mice
(Sarda et al. 2012).
The Hu-FRGTM mice produced a number of human-specific metabolites that had previously been found in vitro
(Kumar et al. 2002), but which were less abundant in Mu-FRGTM, such as the hydroxy-glucuronides (M5, M24,
M25, M33), cysteine conjugates (M19, M21), DCF-hydroxy glucuronide (M35) and an unassigned conjugate
(M42). One minor metabolite, hydroxy-methoxy-DCF (M7 and M11) (Tables 2 and 4) was observed in Hu-
FRGTM urine and may correspond to “metabolite VI” (Faigle et al. 1988; Degen et al. 1988) which was found to
accumulate in human plasma and was also observed in human urine, albeit only in small amounts (ca. 1% dose).
The same metabolite was also observed in the plasma of chimeric, liver humanized, TK-NOG mice
(Kamimura et al. 2010). In the present study, the presence of metabolites M7 and M11 only appeared in urine of
Hu-FRGTM mice and were not observed in blood at this dose. Overall, there were nevertheless significant
qualitative differences in the metabolite profiles between the various models, including evidence of unique
metabolites for the Hu-FRGTM animals. The metabolic fate of DCF in Hu-FRGTM mice using LC-MS did,
however, share some commonality with those of chimeric Mu-FRGTM and wild type C57BL/6J mice (with the
Mu-FRGTM profiles somewhat intermediate between the C57BL/6J and Hu-FRGTM mice).
Differences were also observed between the metabolism of DCF in Hu-FRGTM mice and that reported for
studies in humans (see Table 4). Determining if these novel metabolites detected in the profiles obtained for the
Hu-FRGTM accurately recapitulate human metabolism is complicated by the fact that the studies performed to
understand the metabolism of DCF in humans were performed over 30 years ago. It is therefore perhaps
unsurprising that a number of minor metabolites of DCF were detected in Hu-FRGTM mice that have not been
reported for humans (Table 4). Moreover, some of the differences observed were associated with a range of
novel and relatively minor compounds found in the present study in mice rather than the major known human
metabolites. This suggests that the reinvestigation of the human in vivo biotransformation of DCF, using modern
techniques, might both improve our understanding of the human metabolism of the drug and, if these novel
minor metabolites were shown to be present, validate further the predictive capabilities of the Hu-FRGTM mice.
The characterisation of the biotransformation of DCF in humans using modern LC-MS-based technologies has,
to date, not yet been undertaken.
With respect to the DILI, associated with DCF, both the acyl glucuronide and oxidative metabolic routes have
been advanced as hypothetical mechanisms in the formation of reactive metabolites, and subsequent covalent
binding to proteins detected in both in vitro or in vivo studies (Park et al. 1987; Tang et al. 1999;
Boelsterli 2003; Uetrecht 2003; Aithal et al. 2004; Zhou et al. 2009; Shaw et al. 2010, Pickup et al. 2012,
Hammond et al. 2014). The combination of much higher production of acyl glucuronides together with the
detection of a “human-specific” pattern of glutathione-derived conjugates in the Hu-FRGTM mouse in this study
suggests that this model may have some value in the investigation of in vivo mechanisms of DCF-related human
DILI.
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Tables and Figures
Table 1 Pharmacokinetic parameters for diclofenac in C57BL/6J mice, Mu-FRG™ mice, Hu-FRG™ mice, and
in healthy human male and female subjects.
Blood Hu-
FRG™ mice
Blood Mu-FRG™
mice
Blood C57BL/6J
mice
Plasma healthy
female subjects*
Plasma healthy
male subjects**
Dose (mg/kg) 10 10 10 0.80 0.71
Cmax (µg/mL) 2.43 ± 0.92 3.86 ± 2.31 2.31 ± 0.53 2.0 ± 0.7 0.91 ± 0.16
Tmax (h) 0.25 0.25 0.25 2.5 ± 1.1 1.25 ± 0.58
AUCinf
(µg.h/mL)3.671 4.94 ± 2.93 2.10 ± 0.49 1.67 ± 0.44 1.20 ± 0.19
t1/2 (h) 0.861, 2 0.52 ± 0.03 0.51 ± 0.49 1.8 ± 2.1 ND
Values are mean ± S.D. unless otherwise stated.
*Data taken from Willis et al. 1976. Seven subjects received a single 50 mg oral dose of diclofenac sodium
(enteric-coated tablet). Using the mean weight of the subjects (62.3 ± 8.4 kg), the dose can be calculated as 0.80
mg/kg.
**Data taken from Zhang et al. 2016. Six subjects received a single-dose administration of 50 mg of Voltaflam
(diclofenac potassium, Novartis (India) Ltd., Mumbai, India). Using a nominal weight of 70 kg, the dose can be
calculated as 0.71 mg/kg. ND, no data were provided for the t1/2.
1Calculated using n = 2 mice (one mouse was terminated 4h after dosing)
2Effective half-life was calculated due to evidence of enterohepatic recycling
Table 2 Summary of HPLC and mass spectrometric data obtained for diclofenac and selected (major and
differentiating) metabolites detected in Hu-FRG™ and Mu-FRG™ mouse urine, faeces, bile and blood.
Metabolites found in both humanized and murinized mice are in bold. Those found only in humanized mice are
in normal font, and those found only in murinized mice are in italic font. The complete list of metabolites is
available in the supplementary data (Table S2).
Peak IDtR
(min) AssignmentElemental
composition [M+H]+
Theoretical m/z (35Cl/12C isotope
[M+H]+)
Δm [observed-theoretical m/z] (mDa) Hu-FRGTM
Δm [observed-theoretical m/z] (mDa) Mu-FRGTM
Presence in biofluids
P 42.4 Diclofenac C14H12Cl2N1O2 296.0240 +0.1 0.0 U,B,F,P
M2 7.3 Dihydroxy C14H12Cl2N1O4 328.0138 +0.8 ND U,B
M3 8.2 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.9 -0.3 U,B
M5 9.8 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.6 - U,B
M7 11.4 Hydroxy-methoxy C15H14Cl2N1O4 342.0294 -1.3 - U
M8 13.0 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.2 -0.4 U,B
M9 13.7 Hydroxy glucose C20H22Cl2N1O8 474.0717 0.0 +0.8 U,B
M10 14.5 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +1.3 -0.2 U
M11 15.0 Hydroxy-methoxy C15H14Cl2N1O4 342.0294 -0.5 - U
M12 15.4 Hydroxy mercapturate C19H19Cl2N2O6S 473.0335 +0.5 +0.1 U,B
M13 16.1 Hydroxy glucuronide, indolinone C20H18Cl2N1O8 470.0404 +0.3 0.0 U,B
M15 17.0 Dihydroxy C14H12Cl2N1O4 328.0138 +0.2 +0.1 U,B
M16 17.9 Hydroxy C14H12Cl2N1O3 312.0189 +0.3 +0.8 U
M17 18.4 Dihydroxy C14H12Cl2N1O4 328.0138 +0.1 - U
M18 18.4 Hydroxy ribose C19H20Cl2N1O7 444.0611 +0.4 +0.2 U
M19 18.7 Hydroxy-deschloro, cysteine C17H18Cl1N2O5S 397.0619 0.0 - U,B
M21 19.9 Hydroxy-deschloro, cysteine C17H18Cl1N2O5S 397.0619 +0.5 - U
M24 22.4 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.8 - U
M25 22.6 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.3 - U
M26 23.2 Deschloro, cysteine C17H18Cl2N2O4S 381.0670 - -0.1 U
M27 24.6 Hydroxy glucuronide, indolinone C20H18Cl2N1O8 470.0404 +0.2 +0.2 U
M28 24.8 Hydroxy glucuronide, indolinone (Na+)
C20H17Cl2N1O8Na 492.0223 0.0 +0.1 U
M29 26.2 Hydroxy cysteine C17H17Cl2N2O5S 431.0230 +0.3 -0.5 U
M33 28.6 Hydroxy glucuronide (Na+) C20H19Cl2N1O9Na 510.0329 +0.3 - U,B
M34 29.2 Hydroxy C14H12Cl2N1O3 312.0189 +0.3 +0.4 U,B
M35 29.7 Hydroxy glucuronide (Na+) C20H19Cl2N1O9Na 510.0329 -0.2 0.0 U,B,F
M36 31.7 Hydroxy C14H12Cl2N1O3 312.0189 0.0 0.2 U,B
M37 32.0 Hydroxy C14H12Cl2N1O3 312.0189 -0.2 0.0 U
M38 32.0 Acyl glucuronide C20H20Cl2N1O8 472.0560 -0.5 +0.2 U
M39 31.5 – 33.8
Transacylated hydroxy glucuronide (cluster)
C20H20Cl2N1O9 488.0510 0.0 -0.1 U,B
M40 33.2 Hydroxy taurine C16H17Cl2N2O5S 419.0230 +2.3 -0.1 U,F
M41 36.6 Hydroxy taurine C16H17Cl2N2O5S 419.0230 +1.1 -0.2 U
M42 37.4 Unassigned conjugate ND 470.0340 ND - U,B,F
M43 38.7 Acyl glucuronide (Na+) C20H19Cl2N1O8Na 494.0380 +0.5 +0.4 U
M44 39.6 Acyl glucuronide (Na+) C20H19Cl2N1O8Na 494.0380 +0.1 +1.0 U,B,P
M45 40.6 Acyl glucuronide C20H20Cl2N1O8 472.0560 +0.2 +0.8 U
M4742.4 - 43.3
Transacylated glucuronides (cluster)
C20H20Cl2N1O8 472.0560 +0.1 -1.0 U,B
M48 46.0 Taurine C16H17Cl2N2O4S 403.0281 -0.1 -0.2 U,B,F,P
Key: metabolite detected in biofluid, U urine; B bile; F faeces; B blood. ND not determined
Table 3 Summary of peak heights for direct taurine conjugates (M48) and hydroxytaurines (M40 and M41)
detected in Hu-FRG™ and Mu-FRG™ mouse urine, faeces, bile and blood at 8 and 24 hours after dosing.
Peak heights Taurine M48 46.0 min
OH Taurine M40 33.2 min
OH Taurine M41 36.6 min
Mu-FRG™ 8h 9.97E+05 6.68E+04 7.53E+04
Mu-FRG™ 24h 1.12E+06 6.77E+04 1.04E+05
Hu-FRG™ 8h 1.40E+04 2.24E+03 1.15E+03
Hu-FRG™ 24h 2.58E+04 3.40E+03 1.46E+03
Ratio 8h Mu-FRG™ / Hu-FRG™ 71 30 65
Ratio 24h Mu-FRG™ / Hu-FRG™ 43 20 71
Mean ratio Mu-FRG™ / Hu-FRG™ 57 25 68
Table 4 Comparison of diclofenac and selected (major and differentiating) metabolites observed in excreta from
human ADME studies (and from a human in vitro study), the present study with Hu-FRG™ and Mu-FRG™
mice, and wild type C57BL/6J mice.
Peak ID
Assignment Human Hu-FRG™
Mu-FRG™
C57BL/6J #
P Diclofenac in vivo Stierlin et al. 1979b +++ +++ ++
M2 Dihydroxy ++ + -
M3 Hydroxy glucuronide in vitro Kumar et al. 2002 +++ +++ ++++
M5 Hydroxy glucuronide in vitro Kumar et al. 2002 ++ - -
M7 Hydroxy-methoxy in vivo Faigle et al. 1988 + - -
M8 Hydroxy glucuronide in vitro Kumar et al. 2002 +++ +++ +++
M9 Hydroxy glucose ++ +++ +++
M10 Hydroxy glucuronide in vitro Kumar et al. 2002 + ++ -
M11 Hydroxy-methoxy in vivo Faigle et al. 1988 ++ - -
M12 Hydroxy mercapturate in vivo Poon et al. 2001 ++ ++ -
M13 Hydroxy glucuronide, indolinone ++ ++ -
M15 Dihydroxy in vivo Stierlin et al. 1979b +++ + +
M16 Hydroxy in vivo Stierlin et al. 1979b ++ + -
M17 Dihydroxy ++ - -
M18 Hydroxy ribose + + +
M19 Hydroxy-deschloro, cysteine ++ - -
M21 Hydroxy-deschloro, cysteine ++ - -
M24 Hydroxy glucuronide ++ - -
M25 Hydroxy glucuronide ++ - -
M26 Deschloro, cysteine - + -
M27 Hydroxy glucuronide, indolinone ++ + -
M28 Hydroxy glucuronide, indolinone +++ +++ +
M29 Hydroxy cysteine + ++ -
M33 Hydroxy glucuronide ++ - -
M34 Hydroxy in vivo Stierlin et al. 1979b +++ + ++
M35 Hydroxy glucuronide +++ ++ +
M36 Hydroxy in vivo Stierlin et al. 1979b +++ ++++*
M37 Hydroxy in vivo Stierlin et al. 1979b +++ ++
M38 Acyl glucuronide in vitro Kumar et al. 2002 +++ ++ -
M39 Transacylated hydroxy glucuronide (cluster)
+++ + +
M40 Hydroxy taurine + + ++
M41 Hydroxy taurine + ++ ++
M42 Unassigned conjugate ++ - -
M43 Acyl glucuronide in vitro Kumar et al. 2002 +++ ++ -
M44 Acyl glucuronide (Na+) in vitro Kumar et al. 2002 ++++ ++++ ++*
M45 Acyl glucuronide in vitro Kumar et al. 2002 + ++ -
M47 Transacylated glucuronides (cluster) in vitro Kumar et al. 2002 +++ ++ +
M48 Taurine + +++ ++++
Key: ++++ detected 5 x 105; +++ detected >105; ++ detected >104; + detected >103, in the present study, or equivalent relative measures in the human and mouse studies; - not reported/below level of detection, * single metabolite observed in a human or mouse study may correspond to one or more metabolites observed in the present study. # Sarda et al. 2012, Sarda et al. 2014.
Fig. 1 Blood concentration-time profiles for diclofenac following single oral administration at 10 mg/kg to (a)
Hu-FRGTM mice (n = 3), (b) to Mu-FRGTM mice (n = 3) and (c) C57BL/6J mice (n = 3). Symbols represent
concentration-time profiles from individual animals.
(c)(b)(a)
Hum anize d M o us e Urine D C F 2 4 h p o o l
Time5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 47.50 50.00
%
0
100MS26_OCT05_PK182_MI_U_012_AMF 1: TOF MS ES+
Sum 0.0300Da2.38e6
39.45
29.54
12.82
8.23
9.75
13.59
24.6916.92
18.52 28.5426.81
31.92
31.54 32.71 38.6133.75
42.31
43.28
M3P
M8M9
M48
M47
M44
M35
M28M31
M36M37/38
M39
M42
M43M17M19
M15M5
M34M32/33
Fig. 2 LC-QTOF-MS profiles of diclofenac and its most abundant metabolites in urine 8-24 h following single
oral administration of 10 mg/kg diclofenac to (a) Hu-FRG™ mice and (b) Mu-FRG™ mice.
(a)
(b)
Fig. 3 LC-QTOF-MS profiles of diclofenac and its most abundant metabolites in bile 8 h following single oral
administration of 10 mg/kg diclofenac to (a) Hu-FRG™ mice and (b) Mu-FRG™ mice. The Mu-FRG™ bile
sample suffered from poor chromatography. Due to limited sample volume the analysis could not be repeated.
(b)
(a)
Fig. 4 LC-QTOF-MS profiles of diclofenac and its most abundant metabolites in faeces 8-24 h following single
oral administration of 10 mg/kg diclofenac to (a) Hu-FRG™ mice and (b) Mu-FRG™ mice.
(a)
(b)
NH
NH
OCl
Cl
S
O
O
OH
NH
NH
OCl
Cl
S
O
O
OH
OH
M48, taurine
M40/41, OH taurine (4'-OH shown)
NH
O
OCl
Cl
O
OH OH
OH
O
OH
M38/43/44/45/47, acyl gluc
NH
O
OCl
Cl
O
OH OH
OH
O
OH
OH
M24/25/32/33/35/39, OH acyl gluc (4'-OH shown)
NH
OH
OCl
Cl
NH
OH
OCl
ClOH
M34, 4'-OH
N
OCl
ClO
OOH
OH
OH
O
OHM13/27/28/31, OH indolinone gluc (4'-OH shown)
NH
OH
OCl
ClO
OOH
OH
OH
O
OH
M3, 4’-OH gluc
M36, 5-OH
NH
OH
OCl
Cl OH
M15, 4’-,5-diOH
NH
OH
OCl
Cl OHOH
Diclofenac
NH
OH
OCl
Cl
OH
NH
OH
OCl
Cl
OH
OCH3
NH
OH
OS
ClOH
NH2
OH
O
M16/22/23/37/46, hydroxy (3'-OH shown)
M7/11, hydroxy methoxy (3'-OH, 4'-OMe shown)
M19/21, 4'-OH, 2'-Cys, deschloro
NH
OH
OCl
ClO
OOH
OH
OHOH
NH
OH
OCl
ClO
OOH
OH OH
M9, OH glucose (4'-OH shown)
M18, OH ribose (4'-OH shown)
NH
OH
OOH
Cl
S
NH2
OH
O
M19/21, 2'-OH, 3'-Cys, deschloro
NH
OH
OCl
Cl
S
NH2
OH
OOH
M29, OH cysteine (4'-OH, 3'-Cys shown)
NH
OH
OCl
Cl
S
NH
OH
OOH
O
CH3
M12, OH mercapturate (4'-OH, 3'-NAC shown)
M1/2/17/20, diOH
M4/5/6/8/10, OH gluc
M42, unassigned conjugate
Fig. 5 Proposed metabolic fate of diclofenac in Hu-FRG™ mouse.
NH
NH
OCl
Cl
S
O
O
OH
NH
NH
OCl
Cl
S
O
O
OH
OH
M48, taurine
M14/30/40/41, OH taurine (4'-OH shown)
NH
O
OCl
Cl
O
OH OH
OH
O
OH
M38/43/44/45/47, acyl gluc
NH
O
OCl
Cl
O
OH OH
OH
O
OH
OH
M35/39, OH acyl gluc (4'-OH shown)
NH
OH
OCl
Cl
NH
OH
OCl
ClOH
M34, 4'-OH
N
OCl
ClO
OOH
OH
OH
O
OHM13/27/28/31, OH indolinone gluc (4'-OH shown)
NH
OH
OCl
ClO
OOH
OH
OH
O
OH
M3, 4’-OH gluc
M36, 5-OH
NH
OH
OCl
Cl OH
M15, 4’-,5-diOH
NH
OH
OCl
Cl OHOH
Diclofenac
NH
OH
OCl
Cl
OH
M16/22/23/37/46, hydroxy (3'-OH shown)
NH
OH
OCl
ClO
OOH
OH
OHOH
NH
OH
OCl
ClO
OOH
OH OH
M9, OH glucose (4'-OH shown)
M18, OH ribose (4'-OH shown)
NH
OH
OS
Cl
NH2
OH
O
M26, 2'-Cys, deschloro
NH
OH
OCl
Cl
S
NH2
OH
OOH
M29, OH cysteine (4'-OH, 3'-Cys shown)
NH
OH
OCl
Cl
S
NH
OH
OOH
O
CH3
M12, OH mercapturate (4'-OH, 3'-NAC shown)
M2, diOH
M4/8/10, OH gluc
Fig. 6 Proposed metabolic fate of diclofenac in Mu-FRG™ mouse.
SUPPLEMENTARY DATA
The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG
mice
C.E. Wilsona, A.P. Dickieb, K. Schreiterc, R. Wehrc, E.M. Wilsond, J. Biald, N. Scheere, I.D. Wilsonf, R.J. Rileyg
aNestlé Skin Health R&D, Les Templiers, Route des Colles BP 87, F-06902 Sophia-Antipolis, FrancebEvotec (UK) Ltd, 114 Innovation Drive, Abingdon, Oxfordshire, OX14 4RZ, UK cEvotec International GmbH, Manfred Eigen Campus, Essener Bogen 7, Hamburg, Germany dYecuris Corporation, PO Box 4645, Tualatin OR 97062, USA eCEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Str. 60-62, 51105 Cologne, GermanyfDept. of Surgery and Cancer, Imperial College, London, UKgEvotec (UK) Ltd, Alderley Park, Nether Alderley, Cheshire, SK10 4TG, UK
Table S1 Plasma HSA concentrations determined by ELISA indicating degree of humanization in Hu-FRG™
mice.
Date plasma sampled 16th July 2015(Day -3)
21st July 2015(Day 2)
Animal ID Plasma [HSA] ug/mL#8 5088 8660#9 7346 5356
#10 11588 -#11 9934 5930
#12 11190 8659
#13 12602 9940
#14 5088 8660
N.B. Animal #10 was terminated 4h after dosing. All tested animals presented plasma HSA levels > 4 mg/mL,
i.e. were > 90 % humanized on days 24 and 29 after removal of NTBC diet.
Supplementary Table S2 Full summary table of HPLC and mass spectrometric data obtained for diclofenac
and its metabolites in Hu-FRG™ and Mu-FRG™ mouse urine, faeces, bile and blood. Metabolites found in
both humanized and murinized mice are in bold. Those found only in humanized mice are in normal font, and
those found only in murinized mice are in italic font.
Peak IDtR
(min) AssignmentElemental
composition [M+H]+
Theoretical m/z (35Cl/12C isotope
[M+H]+)
Δm [observed-theoretical m/z] (mDa) Hu-FRGTM
Δm [observed-theoretical m/z] (mDa) Mu-FRGTM
Presence in biofluids
P 42.4 Diclofenac C14H12Cl2N1O2 296.0240 +0.1 0.0 U,B,F,P
M1 6.8 Dihydroxy C14H12Cl2N1O4 328.0138 +0.7 - U
M2 7.3 Dihydroxy C14H12Cl2N1O4 328.0138 +0.8 ND U,B
M3 8.2 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.9 -0.3 U,B
M4 8.8 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +1.0 +0.8 U
M5 9.8 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.6 - U,B
M6 10.1 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.9 - U
M7 11.4 Hydroxy-methoxy C15H14Cl2N1O4 342.0294 -1.3 - U
M8 13.0 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.2 -0.4 U,B
M9 13.7 Hydroxy glucose C20H22Cl2N1O8 474.0717 0.0 +0.8 U,B
M10 14.5 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +1.3 -0.2 U
M11 15.0 Hydroxy-methoxy C15H14Cl2N1O4 342.0294 -0.5 - U
M12 15.4 Hydroxy mercapturate 473.0335 +0.5 +0.1 U,B
M13 16.1 Hydroxy glucuronide, indolinone C20H18Cl2N1O8 470.0404 +0.3 0.0 U,B
M14 16.9 Hydroxy taurine C16H17Cl2N2O5S 419.0230 - +1.1 U
M15 17.0 Dihydroxy C14H12Cl2N1O4 328.0138 +0.2 +0.1 U,B
M16 17.9 Hydroxy C14H12Cl2N1O3 312.0189 +0.3 +0.8 U
M17 18.4 Dihydroxy C14H12Cl2N1O4 328.0138 +0.1 - U
M18 18.4 Hydroxy ribose C19H20Cl2N1O7 444.0611 +0.4 +0.2 U
M19 18.7 Hydroxy-deschloro, cysteine C17H18Cl1N2O5S 397.0619 0.0 - U,B
M20 19.1 Dihydroxy C14H12Cl2N1O4 328.0138 +0.3 - U
M21 19.9 Hydroxy-deschloro, cysteine C17H18Cl1N2O5S 397.0619 +0.5 - U
M22 21.2 Hydroxy C14H12Cl2N1O3 312.0189 +0.7 +0.4 U
M23 21.8 Hydroxy C14H12Cl2N1O3 312.0189 -0.9 +0.2 U
M24 22.4 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.8 - U
M25 22.6 Hydroxy glucuronide C20H20Cl2N1O9 488.0510 +0.3 - U
M26 23.2 Deschloro, cysteine C17H18Cl2N2O4S 381.0670 - -0.1 U
M27 24.6 Hydroxy glucuronide, indolinone C20H18Cl2N1O8 470.0404 +0.2 +0.2 U
M28 24.8Hydroxy glucuronide, indolinone (Na+)
C20H17Cl2N1O8Na 492.0223 0.0 +0.1 U
M29 26.2 Hydroxy cysteine C17H17Cl2N2O5S 431.023 +0.3 -0.5 U
M30 26.3 Hydroxy taurine C16H17Cl2N2O5S 419.0230 - +0.9 U
M31 26.9 Hydroxy glucuronide, indolinone (Na+)
C20H17Cl2N1O8Na 492.0223 +0.6 +0.4 U
M32 28.3 Hydroxy glucuronide (Na+) C20H19Cl2N1O9Na 510.0329 +0.2 - U,B
M33 28.6 Hydroxy glucuronide (Na+) C20H19Cl2N1O9Na 510.0329 +0.3 - U,B
M34 29.2 Hydroxy C14H12Cl2N1O3 312.0189 +0.3 +0.4 U,B
M35 29.7 Hydroxy glucuronide (Na+) C20H19Cl2N1O9Na 510.0329 -0.2 0.0 U,B,F
M36 31.7 Hydroxy C14H12Cl2N1O3 312.0189 0.0 +0.2 U,B
M37 32.0 Hydroxy C14H12Cl2N1O3 312.0189 -0.2 0.0 U
M38 32.0 Acyl glucuronide C20H20Cl2N1O8 472.0560 -0.5 +0.2 U
M3931.5 – 33.8
Transacylated hydroxy glucuronide (cluster)
C20H20Cl2N1O9 488.0510 0.0 -0.1 U,B
M40 33.2 Hydroxy taurine C16H17Cl2N2O5S 419.0230 +2.3 -0.1 U,F
M41 36.6 Hydroxy taurine C16H17Cl2N2O5S 419.0230 +1.1 -0.2 U
M42 37.4 Unassigned conjugate ND 470.0340 ND - U,B,F
M43 38.7 Acyl glucuronide (Na+) C20H19Cl2N1O8Na 494.0380 +0.5 +0.4 U
M44 39.6 Acyl glucuronide (Na+) C20H19Cl2N1O8Na 494.0380 +0.1 +1.0 U,B,P
M45 40.6 Acyl glucuronide C20H20Cl2N1O8 472.0560 +0.2 +0.8 U
M46 41.1 Hydroxy C14H12Cl2N1O3 312.0189 +0.7 +0.3 U
M47 42.4 - 43.3
Transacylated glucuronides (cluster)
C20H20Cl2N1O8 472.0560 +0.1 -1.0 U,B
M48 46.0 Taurine C16H17Cl2N2O4S 403.0281 -0.1 -0.2 U,B,F,P
Key: metabolite detected in biofluid U urine, B bile, F faeces, B blood. ND not determined
Supplementary Fig. S1 Representative extracted ion spectra of diclofenac and its metabolites. Mass spectra
extracted from Hu-FRGTM samples are shown on the top and Mu-FRGTM on the bottom. Metabolites only
present in one strain are annotated with “Hu” or “Mu”. Where a regioisomer is not defined, the primary form
(e.g. 4’-hydroxy, O-β-glucuronide) is shown to represent the class of biotransformation assigned from the
empirical formula of the metabolite. Peaks annotated with “!” have been flagged by the MassLynx software as
not fully resolved from an interference peak.
Diclofenac
m/z285 290 295 300 305
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1226 (42.345)
3.60e5296.0241
294.0085286.1498
298.0215
300.0193
301.0216
M+H+
M+H+
m/z285 290 295 300 305
%
0
100MS26_OCT05_PK182_MI_U_004 1226 (42.345) 1: TOF MS ES+
2.70e5296.0240
294.0100287.2241 289.2524
298.0213
300.0191
303.2322
M1 (Hu)
m/z328 330 332 334 336 338 340
%
0
100
MS26_OCT05_PK182_MI_U_011_AMF 195 (6.752)1.71e4328.0145
330.0121
335.1008
!332.2111
!337.1079 !
339.1285
M+H+
M2
m/z320 325 330 335 340
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 212 (7.327)
2.85e4335.1010
322.1064
328.0146
326.2071
330.0117
!334.1693
!337.1151
338.1173
N.B. Clean spectra could not be obtained in Mu-FRG.
M+H+
M3
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 238 (8.226)
2.33e5488.0519
485.1681
490.0497
491.0533
498.2260493.0493
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_004 241 (8.327) 1: TOF MS ES+
2.47e5488.0507
485.1417480.2289
490.0483
491.0508
493.0494496.2299
M+H+
M+H+
M4
m/z480 485 490 495 500
%
0
100
MS26_OCT05_PK182_MI_U_011_AMF 256 (8.854)1.41e4488.0520
!480.2482
!485.1893
!481.2470
490.0490
!499.1980
!490.2242
!499.1594
m/z487 488
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 256 (8.854)
3.51e3!488.0518
!487.2139
!487.1917!
486.2514
!486.2891
!487.1407
!487.1058
!487.2520
!487.3097
M+H+
M+H+
M5 (Hu)
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 283 (9.787)
1.76e4488.0516
480.1539
486.1436
490.0493
499.2003
490.2757!
497.1847
!497.1494
M+H+
M6 (Hu)
m/z480 481 482 483 484 485 486 487 488 489
%
0
100
MS26_OCT05_PK182_MI_U_011_AMF 290 (10.023)4.30e3485.1713
!483.2040
!482.2235
!488.0519
!486.1786
!;487.1732
489.1668
M+H+
M7 (Hu)
m/z341.900 342.000 342.100 342.200
%
0
100
MS26_OCT05_PK182_MI_U_011_AMF 332 (11.483)1.22e3!
342.1981342.0281
341.9832
!342.1794
!342.1606
!342.1288
M+H+
M8
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 375 (12.957)
1.93e4488.0512
485.1877483.2134
490.0480
490.1985
496.2097491.0532496.2645
499.1792
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_004 376 (12.991) 1: TOF MS ES+
3.75e4488.0506
487.0446
485.2941480.2426
490.0487
491.0533
499.2722496.2523
M+H+
M+H+
M9
m/z465 470 475 480 485
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 395 (13.653)
1.41e5474.0717
473.2314
471.1537467.1665
476.0690
477.0727483.2091
479.0699
m/z465 470 475 480 485
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 397 (13.720)
2.68e5474.0725
473.2322
469.0750
476.0701
477.0727
483.2122
M+H+
M+H+
M10
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 421 (14.552)
4.73e3488.0523
486.1714
484.2316
495.1967!488.2437
!490.1949
!491.0406
!496.2007
!498.2745
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 422 (14.586)
1.37e4488.0508
480.2430
486.1729!
481.2469
!490.3228
491.0528 !495.2019
492.0479 !496.2137
M+H+
M+H+
M11 (Hu)
m/z330 335 340 345 350
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 434 (14.991)
1.82e4342.0289
!335.1876
!;332.1897
!341.1724
344.0269
346.0218!
349.2025
M+H+
M12
m/z465 470 475 480 485
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 443 (15.315)
9.69e3473.0340
469.1751
475.0306
!476.3029
!483.2079!
481.1666
m/z465 470 475 480 485
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 447 (15.451)
4.17e4473.0336
468.2512
475.0319
476.0323
477.1028 !482.1966
M+H+
M+H+
M13
m/z460 462 464 466 468 470 472 474
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 464 (16.026)
3.17e4470.0407
467.1898
463.2560!
464.2563
472.0388
473.1783
474.0339
m/z460 462 464 466 468 470 472 474
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 468 (16.161)
2.55e4470.0404
467.1898463.2514
464.2583
472.0385
473.2767!
474.2828
M+H+
M+H+
M14 (Mu)
m/z414 416 418 420 422 424 426 428
%
0
100
MS26_OCT05_PK182_MI_U_004_AMF 491 (16.958)4.33e3419.0241
415.1894!
415.2597
416.1364
!416.2615
421.0202
!421.1837
428.2103421.7498
!422.2531
!425.1814
M+H+
M15
m/z326 327 328 329 330 331 332 333 334
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 492 (16.992)
1.37e5328.0140
327.0055326.2140
330.0111
329.0135
331.0134 332.0088
332.1593
m/z326 327 328 329 330 331 332 333 334
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 496 (17.147)
2.00e4328.0139
327.0064
326.7084
330.0114
329.0046332.1604
331.0125333.2101
333.2495
M+H+
M+H+
M16
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 519 (17.925)
1.64e4312.0192
308.1269
300.1642303.1688 311.2033
314.0163
315.0211
319.2285
m/z308 310 312 314 316 318 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 521 (17.992)
6.96e3316.1807311.1644
!308.1939
312.0197
315.6889
!314.0155
!317.2090
319.1898
M+H+
M+H+
M17 (Hu)
m/z320 325 330 335 340
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 532 (18.384)
4.31e4328.0139
327.1372!
322.1673
330.0109
!332.0078
!337.2354
M+H+
M18
m/z435 440 445 450 455
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 532 (18.384)
7.68e3444.0615
!439.1869
!435.2444
446.0580
!453.2095
449.1826!
453.1559
m/z435 440 445 450 455
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 535 (18.486)
1.77e4444.0613
!440.2647!
435.2521
!439.1936
!437.1761
443.7659
446.0578
447.0638453.1559
M+H+
M+H+
M19 (Hu)
m/z390 391 392 393 394 395 396 397 398 399
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 541 (18.688)
5.54e4398.2533
397.0619396.1699395.1661!
391.9753
!399.0597
M+H+
M20 (Hu)
m/z320 325 330 335 340
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 553 (19.114)
6.55e3328.0141
324.1204
323.1835
335.0217
330.0103
!333.2392
333.2087
!337.2039
M+H+
M21 (Hu)
m/z390 391 392 393 394 395 396 397 398 399
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 577 (19.926)
7.64e3398.2536
397.0624
393.1954!
391.1930 !393.2267
!395.2036
399.0599
M+H+
M22
m/z304 306 308 310 312 314 316 318 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 612 (21.149)
3.75e3312.0196
304.1709
305.1787 !308.1997
!314.0171
!314.2007
!315.2007
319.1933
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 615 (21.250)
1.82e4312.0193
!308.2084
!307.1965!
303.1692!
303.1974
310.1675
314.0168
314.1428
!316.2124
M+H+
M+H+
M23
m/z310 312 314 316 318 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 627 (21.656)
3.32e3312.0180
!309.1283
!309.1765
!314.0151
!319.2290!
315.1963!
319.1987
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 630 (21.757)
1.37e4312.0191
310.1626305.1797
!303.1690
314.0158
!319.2265317.2093
M+H+
M+H+
M24 (Hu)
m/z488 490 492 494 496 498 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 647 (22.352)
8.22e3488.0518
490.0504
!491.0533
!492.0509
!499.2021
!496.2771
M+H+
M25 (Hu)
m/z488 490 492 494 496 498 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 658 (22.724)
1.03e4488.0513
489.2626
!490.0478
490.2667 !499.1894
!491.0548
!496.2329
!499.2093
M+H+
M26 (Mu)
m/z372 374 376 378 380 382 384 386
%
0
100
MS26_OCT05_PK182_MI_U_004_AMF 671 (23.183)1.01e4381.0669
!372.2608
!375.1934
!377.1175
!380.2258
383.0628
!385.2246
M+H+
M27
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 711 (24.555)
1.62e4470.0406
463.2879!
464.2926
472.0387
473.0400476.2507
m/z460 462 464 466 468 470 472
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 716 (24.744)
2.64e3470.0406
!465.2109
!463.2628
!460.2554
!463.2332
!465.1892
466.2173467.2270
!468.2265
472.0381
M+H+
M+H+
M28
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 719 (24.846)
1.86e5492.0223
486.2332481.1911 487.2377
494.0198
495.0245
497.0232
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 720 (24.879)
2.50e5492.0224
486.2332
480.8027 489.3132
494.0203
495.0233
497.0207
M+Na+
M+Na+
M29
m/z420 425 430 435
%
0
100
MS26_OCT05_PK182_MI_U_004_AMF 762 (26.319)1.25e4431.0225
423.2892
!424.2690
!424.2933
427.2827
433.0195
437.2581
435.0177
437.5932
437.9314
m/z428 429 430 431 432
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 759 (26.218)
983!428.2141
!427.2668
!428.1908
!428.1712
!431.0233
!429.2104
!429.0904
!428.2828
!430.2979
!430.2640
!429.2522
!430.1566
!431.1896
!431.2287
!431.3025
M+H+
M+H+
M30 (Mu)
m/z410 412 414 416 418 420 422
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 762 (26.319)
4.27e3!411.1646 419.0239
!415.2537
!413.2475
418.1681
!419.2289
!421.0141
!421.0278
!422.2623
M+H+
M31
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 776 (26.813)
5.13e4492.0229
486.2321
480.2790
481.2817487.2363
488.2378
494.0201
495.0240
497.0199
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 781 (26.982)
7.94e4492.0227
486.2330
480.2773487.2395
487.6295
494.0201
495.0232
496.3080
M+Na+
M+Na+
M32 (Hu)
m/z500 505 510 515 520
%
0
100
MS26_OCT05_PK182_MI_U_011_AMF 820 (28.320)3.55e3510.0331
!501.2656
501.6372 !507.3138
512.0332
!514.0330 516.3104
516.6473
!517.3231
M+Na+
M33 (Hu)
m/z500 505 510 515 520
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 829 (28.644)
1.12e4510.0332
!507.3078!
505.2368
512.0336
513.2650
!514.0261 !
519.2845
M+Na+
M34
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 847 (29.253)
9.66e4312.0192
303.1702310.2012305.1844
314.0162
315.0194
319.1872
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 852 (29.422)
4.03e4312.0193
306.1757303.1706
309.1916
314.0169
315.0181
316.1591
M+H+
M+H+
M35
m/z500 505 510 515 520
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 858 (29.645)
2.79e5510.0327
503.2283 509.9364
512.0303
513.0335
515.0295
m/z500 505 510 515 520
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 865 (29.881)
4.56e4510.0329
507.3084503.3031
507.2782
516.3101512.0313
514.0292
516.6446
516.9784
517.3134
519.2744
M+Na+
M+Na+
M36
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 916 (31.645)
2.83e5312.0189
310.2013
303.1713 310.0042
314.0162
316.0138
318.1720
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 922 (31.848)
1.67e5312.0191
303.1715 311.0113
304.1717
314.0169
316.0138316.1579
M+H+
M+H+
M37
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 920 (31.781)
6.39e4312.0187
303.1711 310.2011
310.0042304.1736
314.0158
316.0145
319.2259
m/z300 305 310 315 320
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 929 (32.085)
6.05e4312.0189
307.1925
303.1697 310.2020
314.0168
316.0128
319.2273
M+H+
M+H+
M38
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 922 (31.848)
1.49e5472.0555
470.0408
464.2812 469.2399
474.0536
475.0568
477.0533
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 934 (32.254)
1.02e5472.0562
463.2460
470.3114464.2505
470.0407466.2469
474.0534
475.1718
476.3074
M+H+
M+H+
M39
m/z480 485 490 495 500
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 914 (31.578)
5.54e4488.0510
480.2809483.2140
490.0486
491.0505
493.2431 497.1238
m/z480 482 484 486 488 490
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 982 (33.916)
6.23e3488.0509
487.3029480.2964
485.2957481.2993 483.2640
482.2938 485.3284
488.2502
M+H+
M+H+
M40
m/z416 418 420 422 424 426 428 430
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1058 (36.546)
1.15e3419.0253
!415.0098
!416.2597
421.0237 !428.3025
427.1799
!423.1904
!423.2191
m/z410 415 420 425 430
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 967 (33.409)
4.98e4419.0229
411.3248 415.2533
421.0197
429.3353
423.0185 428.2983
M+H+
M+H+
M41
m/z410 412 414 416 418 420
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 962 (33.220)
1.67e3!419.0241
!415.2500!
413.2288!
410.2907
!413.1993
!412.2717
414.2271
!416.2496
!419.0100
!417.2222
!421.0164
!420.0263
m/z410 415 420 425 430
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1061 (36.647)
7.53e4419.0228
411.2904414.2923
421.0199
423.0164
428.3004
M+H+
M+H+
M42 (Hu)
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1082 (37.377)
1.87e4470.0340
!461.2314
464.3213
466.3146
472.0318
474.0262 !476.3065
M+H+
Structure not assigned
M43
m/z485 490 495 500 505
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1117 (38.580)
2.20e4494.0385
490.2660
487.3056
496.0359
497.0397
498.0349
502.2562
m/z485 490 495 500 505
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1122 (38.749)
1.13e5494.0384
490.2641
488.2474
496.0362
498.0338
504.2867499.0368
M+Na+
M+Na+
M44
m/z485 490 495 500 505
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1148 (39.648)
1.30e6494.0381
493.9828490.2647
496.0355
497.0385
499.0366504.2626
m/z485 490 495 500 505
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1151 (39.749)
1.47e6494.0390
490.2657486.0670
496.0365
497.0397
499.0381 503.2750
M+Na+
M+Na+
M45
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1176 (40.615)
1.26e4472.0562
!463.2014 466.9984
474.0526
!476.2950
!477.3016
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1179 (40.716)
1.38e4472.0568
460.2561
467.0016!461.2528 468.9958
474.0547 !476.2911
479.2758
M+H+
M+H+
M46
m/z300 302 304 306 308 310 312 314 316
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1189 (41.074)
6.80e3312.0196
!304.1578
!311.0143
314.0168
!314.2323
!316.1183
m/z300 302 304 306 308 310 312 314 316
%
0
100MS26_OCT05_PK182_MI_U_004 1189 (41.074) 1: TOF MS ES+
4.98e4312.0192
311.0118!305.2507
!300.2109
314.0168
316.0154
M+H+
M+H+
M47
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1233 (42.582)
3.30e5472.0561
472.0070460.0434 464.2845
474.0534
475.0565
477.0528
m/z460 465 470 475 480
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1235 (42.649)
4.50e5472.0550
471.0521460.0484463.2653
474.0529
475.0558
477.0513
M+H+
M+H+
M48
m/z395 400 405 410 415
%
0
100MS26_OCT05_PK182_MI_U_011_AMF 1334 (46.076)
1.08e4403.0280
!401.2225
!398.2947
405.0259
412.2201!
406.0320
!411.2950
412.3057!;414.2771
m/z395 400 405 410 415
%
0
100MS26_OCT05_PK182_MI_U_004_AMF 1333 (46.042)
5.12e5403.0279
402.9713398.2999
405.0249
407.0230
408.0252411.2898
M+H+
M+H+