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

claire.wilson@galderma.com; claire.wilson1000@gmail.com

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+