species differences in the oxidative desulfuration of a...

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Species Differences in the Oxidative Desulfuration of a Thiouracil-Based

Irreversible Myeloperoxidase Inactivator by Flavin-Containing

Monooxygenase Enzymes

Heather Eng, Raman Sharma, Angela Wolford, Li Di, Roger B. Ruggeri, Leonard Buckbinder,

Edward L. Conn, Deepak K. Dalvie, and Amit S. Kalgutkar

Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, CT

(H.E., R.S., A.W., L.D.), La Jolla, CA (D.K.D.) and Cambridge MA (A.S.K.); Worldwide

Medicinal Chemistry (E.L.C., R.B.R.); Cardiovascular and Metabolic Research Unit,

Cambridge, MA (L.B.)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 14, 2016 as DOI: 10.1124/dmd.116.070185

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Running Title: Oxidative desulfuration by flavin-containing monooxygenases

Address correspondence to: Amit S. Kalgutkar, Pharmacokinetics, Dynamics, and

Metabolism-New Chemical Entities, Pfizer Worldwide Research and Development, 610 Main

Street, Cambridge, MA 02139, USA. Tel: +(617)-551-3336. E-mail: [email protected]

Text Pages (including references): 30

Tables: 1

Figures: 8

References: 59

Abstract: 252

Introduction: 471

Discussion: 1550


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Abbreviations used are: 1, 6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-

dihydropyrimidin-4(1H)-one; AUC(0-∞), area under the plasma concentration–time curve from

zero to infinity; CID, collision-induced dissociation; CLint, intrinsic clearance; CLp, plasma

clearance; CLrenal, renal clearance; DMSO, dimethyl sulfoxide; FMO, flavin-containing

monooxygenase; GSH, reduced glutathione; H2O2, hydrogen peroxide; LC-MS/MS, liquid

chromatography tandem mass spectrometry; KM, Michaelis-Menten constant; M1, 5-(2,4-

dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one; MPO, myeloperoxidase;

NADPH, reduced nicotinamide adenine dinucleotide phosphate; P450, cytochrome P450; PTU,

propylthiouracil; SAR, structure-activity relationship; t1/2, half-life; tR, retention time; TPO,

thyroid peroxidase; Vdss, steady state distribution volume; Vmax,maximum rate of oxidative

desulfurization.


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Abstract:

N1-Substituted-6-arylthiouracils (represented by 6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-

thioxo-2,3-dihydropyrimidin-4(1H)-one (1)) represent a novel class of selective irreversible

inhibitors of human myeloperoxidase. The present account represents a summary of our in vitro

studies on the facile oxidative desulfuration in 1 to a cyclic ether metabolite M1 in NADPH-

supplemented rat (t1/2 (mean±standard deviation)=8.6±0.4 min) and dog liver microsomes

(t1/2=11.2±0.4 min), but not in human liver microsomes (t1/2 >120 min). The in vitro metabolic

instability also manifested in moderate-to-high plasma clearances of the parent compound in rats

and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of

liver microsomes or co-incubation with the flavin-containing monooxygenase (FMO) inhibitor

imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of 1 to M1

was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations

with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally

catalyzed the conversion of 1 to M1. FMO1 is not expressed in adult human liver, which

rationalizes the species difference in oxidative desulfuration. Oxidation by FMO1 followed

Michaelis-Menten kinetics with KM, Vmax, and CLint values of 209 μM, 20.4 nmoL/min/mg

protein, and 82.7 μL/min/mg protein, respectively. Addition of excess glutathione essentially

eliminated the conversion of 1 to M1 in NADPH-supplemented rat and dog liver microsomes,

which suggested that the initial FMO1-mediated S-oxygenation of 1 yields a sulfenic acid

intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion

or undergoing further oxidation to a more electrophilic sulfinic acid species that is trapped

intramolecularly by the pendant alcohol motif in 1.


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Introduction

We recently reported structure-activity relationship (SAR) studies on N1-substituted-6-aryl-2-

thiouracil derivatives as irreversible, mechanism-based inactivators of the hemoprotein

myeloperoxidase (MPO, EC 1.11.2.2) with a high degree of selectivity for MPO relative to

peroxidases such as thyroid peroxidase (TPO) and cytochrome P450 (P450) enzymes (Ruggeri et

al., 2015). The thiouracil analogs behave as suicide substrates of MPO and covalently adduct to

the heme prosthetic group through an oxidized sulfur species (presumably a thiyl radical)

generated during catalysis (Ruggeri et al., 2015; Tidén et al., 2011). The antithyroid drug

propylthiouracil (PTU, Figure 1), which irreversibly inhibits MPO and TPO in a non-selective

fashion (Lee et al., 1990; Ruggeri et al., 2015), was used as a starting point in our SAR work to

identify selective MPO inhibitors. Introduction of polar N1 substituents and replacement of the

C6 propyl group in PTU with electron-rich aromatic functionalities resulted in significant

improvements in MPO inhibitory activity (inferred from inactivation kinetics parameters (kinact

and KI) and partition ratio) and virtually abolished TPO inhibition with the resultant compounds.

Concern over the risk of immune-mediated toxicity (e.g., agranulocytosis and hepatotoxicity)

associated with chronic PTU treatment (Cooper, 2005; Futcher and Massie, 1950; Ichiki et al.,

1998) via oxidative bioactivation of the thiouracil motif to protein- and thiol-reactive

intermediates (Jiang et al., 1994; Lee et al., 1988, Lee et al., 1990; Waldhauser and Uetrecht,

1991) was principally mitigated by tethering pendant nucleophilic groups to the N1-substituent,

which could potentially quench reactive species in an intramolecular fashion. Out of this

exercise emerged the lead compound 6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-

dihydropyrimidin-4(1H)-one (1, Figure 1) with significant improvements noted in MPO


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inactivation potency and selectivity relative to PTU. Intramolecular trapping of reactive species

was demonstrated by reacting 1 with excess hydrogen peroxide (H2O2) (Kalm, 1961; Kitamura,

1934), which quantitatively converted 1 to the pharmacologically inactive cyclic ether 5-(2,4-

dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one (M1, Figure 1), presumably

via an unstable oxidized sulfur intermediate (Ruggeri et al., 2015). Importantly, no thiol

conjugates of 1 were generated upon addition of reduced glutathione (GSH) to the H2O2 and

MPO/H2O2 incubations. Moreover, compound 1 was resistant towards metabolic turnover in

reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver

microsomes (half-lives (t1/2) > 120 min) and cryopreserved human hepatocytes (t1/2 > 240 min),

which was generally consistent with its physicochemical properties (molecular weight = 308,

lipophilicity (logD7.4 = 1.1), and topological polar surface area = 71.03 Å2). In contrast, a high

metabolic turnover of 1 was noted in NADPH-supplemented rat and dog liver microsomes,

which translated in moderate to high plasma clearance (CLp) in these preclinical species. In vitro

mechanistic studies were initiated to rationalize the species difference in metabolism and

revealed a facile conversion of 1 principally to the cyclic ether metabolite M1 by rat and dog

liver flavin-containing monooxygenase (FMO) 1, which is not expressed in adult human liver.

The collective findings from these studies are reported, herein.

Materials and Methods

Materials. The synthesis of compound 1 (chemical purity > 99% by HPLC and NMR) has been

previously reported (Ruggeri et al., 2015). The preparation of the M1 metabolite is described in

the supplemental section. NADPH, 1-aminobenzotriazole, reduced GSH, and imipramine were

purchased from Sigma-Aldrich (St. Louis, MO). Pooled male Wistar-Han rat and male and


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female human liver microsomes (pool of 126 livers, age 8-10 weeks) were purchased from BD

Gentest (Woburn, MA), male beagle dog liver microsomes (pool of 14 livers, age 1-4 years) and

pooled male and female human kidney microsomes from XenoTech (Kansas City, KS).

Recombinant human FMO1, FMO3, and FMO5 supersomes were purchased from Corning

(Oneonta, New York).

Liver and Kidney Microsomal Stability. Stock solutions of 1 were prepared in dimethyl

sulfoxide (DMSO) then diluted with methanol and acetonitrile. The final concentration of

solvents in the incubation mixture were 0.025% DMSO, 0.5% methanol, and 0.475%

acetonitrile. Assessment of t1/2 in liver or kidney microsomes was determined in triplicate with

microsomes (1 mg/mL microsomal protein for rat, dog, and human) in 0.1�M potassium

phosphate buffer (pH 7.4) containing 3.3 mM MgCl2 at 37�°C. The reaction mixture was

prewarmed with 1.3 mM NADPH at 37�°C for 5�min before initiating the reaction with the

addition of 1 (1 µM). Aliquots of the reaction mixture at 0.25, 5.0, 10, 20, 30, 40 and 60�min

were added to acetonitrile containing 0.1% formic acid and an internal standard terfenadine (2

ng/mL). The samples were centrifuged prior to dilution of supernatant with an equal volume of

water containing 0.1% formic acid and liquid chromatography/tandem mass spectrometry (LC-

MS/MS) analysis of the disappearance of 1 and the formation of cyclized metabolite M1. For

control experiments, NADPH was omitted from these incubations. A parallel incubation of liver

microsomes from rat and dog containing compound 1 (1 μM), GSH (5 mM) and NADPH (1.3

mM) was conducted in order to evaluate redox cycling of the initial S-oxygenation product of 1

formed during the process of enzymatic oxidation. To test the involvement of the thermally

unstable FMO in the oxidation, rat and dog liver microsomes were preincubated at 50 °C for 5


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min in the absence of NADPH co-factor and then cooled on ice followed by incubations

described above. For the purposes of metabolite identification studies, the concentration of 1 in

the microsomal incubations was raised to 10�µM. The duration of the incubation time was

60�min. Separate incubations of 1 (10 μM) were also conducted in human liver microsomes

containing NADPH (1.3 mM) and glutathione (1 mM) for the purposes of trapping potentially

reactive species arising from the oxidative metabolism of 1. To determine the effects of P450 or

FMO inhibition on the metabolic conversion of 1 to M1, 1-aminobenzotriazole (1 mM final

concentration) [P450 inactivator] or imipramine (250 μM final concentration) [FMO competitive

inhibitor] were preincubated with rat and dog liver microsomes in the presence of NADPH for

20 or 2 min, respectively, prior to initiation of the reaction with 1.

Incubations of 1 with Recombinant FMO Isoforms. Potassium phosphate buffer (0.1 M, pH

7.4), magnesium chloride (3.3 mM), NADPH (1.3 mM), and recombinant FMO (0.5 mg/mL)

were combined and pre-warmed at 37 °C for 2 min. To initiate reaction, 1 was added at a final

concentration of 1 µM (final 0.025% DMSO, 0.5% methanol, and 0.475% acetonitrile). At each

time point (0.25, 5.0, 10, 20, 30, 40, and 60 min) a 50 µL aliquot of reaction mixture was

transferred to 200 µL acetonitrile containing 0.1% formic acid and internal standard terfenadine

(2 ng/mL). After centrifugation at 2000 g, equal volumes of supernatant and water containing

0.1% formic acid were mixed, and the disappearance of 1 and formation of M1 was examined by

LC-MS/MS.

To evaluate linearity of product (i.e., M1) formation by human FMO1 and FMO3 isoforms,

recombinant FMO1or FMO3 supersomes (0.1-1 mg/mL) and NADPH (3.3 mM) were


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preincubated in 0.1 M phosphate buffer pH 7.4 for 2 minutes at 37 °C. Reactions (5000 μL)

were initiated by the addition of compound 1 (1 μM), and were allowed to continue at 37 °C for

0.25 to 60 min. Aliquots (50 µL) of reactions were quenched with 200 µL of acetonitrile

containing 0.1% formic acid and internal standard terfenadine (2 ng/mL), and after centrifugation

at 2000 g, supernatants were combined with an equal volume of water containing 0.1% formic

acid, and the formation of M1 was examined by LC-MS/MS.

For determination of the Michaelis-Menten constant KM, maximum rate of oxidative

desulfurization Vmax, and intrinsic clearance (CLint, Vmax/KM) for 1, incubations were repeated at a

single protein concentration and time point determined to be in the linear range of metabolite

formation (0.1 mg/mL FMO1, 1 mg/mL FMO3, 60 min), containing 12 concentrations of 1 (1-

300 µM). Kinetic parameters were obtained for FMO1 using the Michaelis-Menten nonlinear

regression in GraphPad PRISM (La Jolla, CA). As the FMO3 reaction was not saturated within

the range of substrate concentrations tested, the slope of formation rate of M1 versus substrate

concentration (CLint) was calculated using linear regression.

Animal Pharmacokinetics. Dog experiments were conducted in our AAALAC-accredited

facilities and were reviewed and approved by Pfizer Institutional Animal Care and Use

Committee. Rat studies were done at BioDuro, Pharmaceutical Product Development Inc.

(Shanghai, PRC); animal care and in vivo procedures were conducted according to guidelines

from the BioDuro Institutional Animal Care and Use Committee. Jugular vein cannulated male

Wistar-Han rats (~250 g), obtained from Vital River (Beijing, China), and male Beagle dogs (~

8-11 kg) were used for these studies. Rats were provided ad libitum access to water and food.


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Dogs were fasted overnight and fed 4 hr after dosing. Compound 1 was administered

intravenously (i.v.) in 5% DMSO/95% of 30% 2-hydroxypropyl-β-cyclodextrin or 25% 2-

hydroxypropyl-β-cyclodextrin/75% 100 mM Tris buffer (pH 8.0) via the tail vein of rats (n=3) or

saphenous vein (dogs, n=3) at a dose of 1.0 mg/kg in a dosing volume of 1 (rat) or 0.5 (dog)

mL/kg. Serial blood samples were collected before dosing and 0.033 (rat only), 0.083, 0.25, 0.5,

1, 2, 4, 7, and 24 h after dosing. Blood samples from the pharmacokinetic studies were

centrifuged to generate plasma. All plasma samples were kept frozen until analysis. Urine

samples (0–7.0 and 7.0–24 h) were also collected after i.v. administration. Aliquots of plasma or

urine (10-50 μl) were transferred to 96-well blocks and methanol/acetonitrile (1:1, v/v, 200 μl)

containing an internal standard was added to each well. Supernatant was diluted 20-fold with

methanol/water (1:1, v/v) containing 0.1% formic acid. Samples were then analyzed by LC-

MS/MS and concentrations of 1 and M1 in plasma and urine were determined by interpolation

from a standard curve. Range of quantitation was 1-2000 ng/mL for rat (linear R2 0.991 1 and

linear R2 0.996 M1) and 1-5000 ng/mL for dog (linear R2 0.995 1 and quadratic R2 0.978 M1).

Determination of Pharmacokinetic Parameters. Pharmacokinetic parameters were

determined using noncompartmental analysis (Watson v.7.4, Thermo Scientific, Waltham, MA).

The area under the plasma concentration-time curve from t = 0 to 24 h (AUC0-24) and t = 0 to

infinity (AUC0-∞) was estimated using the linear trapezoidal rule and CLp was calculated as the

intravenous dose divided by AUC0–∞i.v.. The terminal rate constant (kel) was calculated by a

linear regression of the log-linear concentration-time curve, and the terminal elimination t1/2 was

calculated as 0.693 divided by kel. Apparent steady state distribution volume (Vdss) was

determined as the i.v. dose divided by the product of AUC0–∞ and kel. Percentage of unchanged 1


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excreted in urine over 24 h was calculated using the following equation: amount (in mg) of 1 in

urine over the 24 h interval post dose/actual amount of the dose of 1 administered (mg) x 100%.

The renal clearance (CLrenal) was derived as the ratio of amount (in mg) of 1 in urine over the 24

h interval post dose/AUC0-24.

LC-MS/MS Analysis for Quantitation of 1 and M1. Concentrations of analytes from in vitro

and in vivo studies were determined on a Sciex 5500 LC-MS/MS triple quadrupole mass

spectrometer (Sciex, Framingham, MA). Analytes were chromatographically separated using

Agilent 1290 (Santa Clara, CA) or Shimadzu LC-20AD (Shimadzu Scientific Instruments, MD)

pumps. A CTC PAL autosampler was programmed to inject 1 or 10 μL on a Phenomenex

Kinetex C18 30 x 3 mm HPLC (Phenomenex, Torrance, CA) or Mac Mod Halo C18 50 x 2.1

mm UPLC column (Mac Mod Analytical, Chadds Ford, PA) using a mobile phase consisting of

water containing 0.1% (v/v) formic acid (solvent A) and acetonitrile containing 0.1% formic

(solvent B) at a flow rate of 0.5 mL/min. Compounds 1 and M1 were detected using

electrospray ionization (positive ion mode) in the multiple reaction monitoring mode monitoring

for mass-to-charge (m/z) transition 309.1 → 164.2 or 291.1 and 275.1 →189.1, respectively.

Compound 1 and M1 standards were fit by least-squares regression of their areas to a weighted

linear equation, from which the unknown concentrations were calculated. The dynamic range of

the assay was 1.0-2000 ng/mL. Assay performance was monitored by the inclusion of quality

control samples with acceptance criteria of ± 30% target values.

Bioanalytical Methodology for Metabolite Identification. Qualitative assessment of the

metabolism of 1 was conducted using a Thermo Finnegan Surveyor photodiode array plus


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detector, Thermo Acela pump and a Thermo Acela Autosampler (Thermo Scientific, West Palm

Beach, FL). The monitoring wavelength (λ) was 280 nm. Chromatography was performed on a

Phenomenex Hydro RP C18 (4.6 mm x 150 mm, 3.5 μm) column. The mobile phase composed

of 5 mM ammonium formate buffer with 0.1% formic acid (pH=3) (solvent A) and acetonitrile

(solvent B) at a flow rate of 1 mL/min. The binary gradient was as follows: solvent A to solvent

B ratio was held at 95:5 (v/v) for 3 min and then adjusted to 55:45 (v/v) from 0 to 35 min, 30:70

(v/v) from 35 to 45 min, and 5:95 (v/v) from 45 to 52 min where it was held for 3 min and then

returned to 95:5 (v/v) for 6 min before next analytical run. Identification of the metabolites was

performed on a Thermo Orbitrap mass spectrometer operating in positive ion electrospray mode.

The spray potential was 4 V and heated capillary was at 275 °C. Xcalibur software version 2.0

was used to control the HPLC-MS system. Product ion spectra were acquired at a normalized

collision energy of 65 eV with an isolation width of 2 amu. Metabolites from liver microsomes

were identified in the full-scan mode (from m/z 100 to 850) by comparing t = 0 samples with t =

60 min samples or through comparison with synthetic standard(s), and structural information was

generated from collision-induced dissociation (CID) spectra of protonated molecular ions.

Results

Microsomal stability. To examine microsomal stability, compound 1 was incubated in rat, dog,

and human liver microsomes or human kidney microsomes in the presence and absence of

NADPH co-factor; periodically, aliquots of the incubation mixture were examined for the

depletion of 1 (Supplemental Figure 1). The t1/2 (mean ± standard deviation) for depletion of 1 in

NADPH-supplemented rat, dog, and human liver microsomes was 8.6 ± 0.4, 11.2 ± 0.4 min, and


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> 120 min, respectively. No substrate depletion (t1/2 > 120 min) was noted in rat, dog, and

human incubations that lacked NADPH co-factor or liver microsomes. Figure 2 depicts the

extracted ion chromatograms of incubation mixtures of 1 in NADPH-supplemented liver

microsomes from rat, dog, and human. The major metabolite M1 was detected in rat and dog

liver microsomes in a NADPH-dependent fashion. The MS2/MS3 spectra of compound 1

(retention time (tR) = 11.25 min, exact mass (M+H)+ = 309.0904) and M1 (tR = 9.61 min, exact

mass (M+H)+ = 275.1026 ) are shown in Supplemental Figures 2 and 3, respectively.

Theoretical exact masses for the proposed fragment ion structures in the CID spectrum of 1 and

M1 were consistent with the observed accurate masses (< 2 ppm difference). The tR and mass

spectrum of M1 was identical to the one discerned with an authentic standard, which was

chemically synthesized via S-methylation of 1 to the corresponding thioether derivative 2

followed by peroxide-mediated oxidative desulfuration/intramolecular cyclization presumably

via an electrophilic sulfoxide intermediate (see supplemental section for detailed synthetic

protocol). Metabolites M2 (tR = 9.19 min) and M3 (tR = 8.75 min) were rat-specific metabolites

with an identical exact mass (295.0747 (M+H)+) and CID spectra (see Supplemental Figure 4 for

a representative CID spectra of M3), implying that these metabolites were isomeric phenols

derived from cytochrome P450-mediated O-demethylations in 1. Consistent with the metabolic

stability results, M1–M3 were only detected in trace quantities in human liver microsomal

incubations of 1 in the presence of NADPH. Compound 1 (1 μM) appeared to be stable (t1/2 >

120 min) towards metabolic turnover in NADPH-supplemented human kidney microsomes, with

minimal amount of M1 (80 nM) formed during the course of the 60 min incubation.


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Compound 1 was devoid of reactive metabolite formation in NADPH-supplemented rat, dog,

and human liver microsomes as inferred from the lack of GSH conjugates formed when GSH (5

mM) was included in the microsomal incubations (data not shown). Incidentally, inclusion of

excess GSH significantly attenuated oxidative desulfuration of 1 (to M1) in liver microsomal

incubations from rat and dog (Supplemental Figure 5). In the presence of GSH, 1 was virtually

resistant to metabolic turnover in NADPH-supplemented dog liver microsomes (t1/2 (- GSH) =

11.2 ± 0.4 min; t1/2 (+ GSH) > 120 min). In contrast, the impact of GSH on the overall metabolic

stability of 1 in NADPH-supplemented rat liver microsomes was less severe (t1/2 (- GSH) = 8.6 ±

0.4; t1/2 (+ GSH) 49.7 ± 1.7 min). Qualitative examination of metabolite formation in NADPH-

and GSH-supplemented rat liver microsomal incubations of 1 (10 μM) revealed that the

formation of the O-demethylated metabolites M2 and M3 was not impacted in the presence of

the thiol nucleophile, which was in contrast to the complete disappearance of M1 (Figure 3).

Identification of enzymes responsible for oxidative desulfuration of 1 to M1. Incubations

of 1 (1 μM) in NADPH-supplemented rat or dog liver microsomes, which had been subjected to

heat treatment (50 °C) for 5 min in the absence of NADPH co-factor induced metabolic

resistance in 1 virtually abrogated the formation of M1 as shown in a representative plot of an

incubation mixture of 1 in heat-inactivated rat liver microsomes (Figure 4), implying a potential

role for a FMO isoform(s) in oxidative desulfuration.

Incubations of 1 (1 μM) in 0.5 mg/mL human recombinant FMO1, FMO3, and FMO5 in the

presence of NADPH revealed that FMO1 was principally responsible for the formation of M1

with a minor contribution from FMO3 (Figure 5). Contribution of FMO5 towards M1 formation


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was insignificant in this analysis; as such enzyme kinetics experiments were not pursued. The

reactions with FMO1 and FMO3 were linear as a function of incubation time (5–60 min) and

protein concentration up to 1.0 mg/mL of microsomal protein (results not shown). The effects of

substrate concentrations on oxidative desulfuration of 1 by recombinant human FMO1 and

FMO3 were investigated, and the results are shown in Figure 6. Oxidation of 1 to M1 by FMO1

followed Michaelis– Menten kinetics (Figure 6, panel A), with Km, Vmax, and CLint ± standard

error values of 209 ± 12 μM, 20.4 ± 0.6 nmoL/min/mg protein, and 97.7 μL/min/mg protein,

respectively. In the case of FMO3, the Michaelis–Menten plot (see Figure 6, panel B) showed

that conversion of 1 to M1 was linear up to the highest substrate concentration of 300 μM,

indicating that the apparent KM value was > 300 μM. Therefore, KM and Vmax values for FMO3

could not be determined. The corresponding CLint value calculated from the slope of the

Michaelis–Menten plot was 0.54 μL/min/mg protein.

Consistent with these observations, conversion of 1 to M1 in NADPH-supplemented rat and

dog liver microsomes was strongly inhibited upon co-incubation with imipramine (250 μM), a

selective inhibitor of FMO1 (Dixit and Roche, 1984; Lee et al., 2009; Yamazaki et al., 2014), as

reflected from changes in t1/2 from 8.6 to ~ 90 min (rats) and 11 to 81 min (dogs). In contrast, the

effects of the non-selective P450 inactivator, 1-aminobenzotriazole (1 mM) (Boily et al., 2015;

Caldwell et al., 2005; Parrish et al., 2015; Strelevitz et al., 2006), on oxidative desulfuration was

less severe (rat: t1/2 from 8.6 to 25 min, dog: t1/2 from 11.0 to 12 min (Figure 7).

Intravenous pharmacokinetics of 1 after single doses to rats and dogs. The pharmacokinetic

parameters describing the disposition of 1 and M1 after administration of 1 to Wistar-Han rats


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and Beagle dogs are shown in Table 1 and Supplemental Figure 6. Compound 1 demonstrated

moderate to high CLp (rat CLp = 73 ± 13 mL/min/kg; dog CLp = 12 ± 3 mL/min/kg), and a

moderate Vdss (rat Vdss = 1.4 ± 0.5 L/kg; dog Vdss = 1.3 ± 0.3 L/kg) resulting in terminal

elimination t1/2 values of 0.4 ± 0.2 and 5.3 ± 0.8 h, respectively, in rats and dogs. M1 was also

detected in circulation after i.v. administration of 1 to rats and dogs. The corresponding AUC0-∞

values of 1 and M1 in rats were 233 ± 37 and 49.8 ± 11.2 ng.h/mL, respectively, whereas, the

corresponding AUC0-∞ values of 1 and M1 in dogs were 1450 ± 310 and 769 ± 136 ng.h/mL,

respectively. M1 had a slightly longer elimination t1/2 (relative to 1) in rats and dogs. Renal

excretion of unchanged 1 (< 1% in rats and ~ 1.1% in dogs) and unchanged M1 (~ 4.7% in rats

and ~ 14.2% in dogs) was relatively low.

Discussion

Concerns over the liberation of indiscriminate electrophilic species during MPO-mediated

oxidation of N1-substituted-6-aryl-2-thiouracils were minimized by ensuring a high partition

ratio for MPO inactivation and by tethering nucleophilic functional groups in proximity of the

thiouracil sulfur. Our medicinal chemistry strategy was also weighted towards the design of

compounds in the lower range of lipophilicity (logD < 1.5, topological polar surface area < 100

Å2) to minimize the potential for oxidative metabolism/bioactivation of the thiouracil motif in

human liver. Apart from peroxidases, enzymatic bioactivation of thioureas and related analogs

(e.g., thiones, thiocarbamides, etc.) to electrophilic intermediates by mammalian P450 and/or

FMO isoforms can also lead to toxicity (Decker and Doerge, 1992; Henderson et al., 2004; Ji et

al., 2007; Neal and Halpert, 1982; Onderwater et al., 1999; 2004; Poulsen et al., 1979; Smith and

Crespi, 2002). For instance, cases of clinical hepato- and/or nephrotoxicity noted with the


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antithyroid drug methimazole (Martinez-Lopez et al., 1962) and the antiparasitic agent

thiabendazole (Manivel et al., 1987) have been causally linked with their metabolism to the

proximal toxicants N-methylthiourea and thioformamide, respectively, via an initial P450-

catalyzed oxidative ring scission of the 2-mercaptobenzimidazole and thiazole motifs present in

these drugs. S-Oxidation of the N-methylthiourea and thioformamide metabolites to reactive

metabolites by FMO enzymes is believed to represent the key step resulting in toxicity (Mizutani

et al., 1993; 2000).

Consistent with our design philosophy, the N1-substituted-6-aryl-2-thiouracil class of MPO

inhibitors (represented in the present study by compound 1) were stable towards metabolism in

NADPH-supplemented human liver microsomes and/or cryopreserved human hepatocytes

(Ruggeri et al., 2015), and were latent to the formation of reactive species as judged from the

absence of GSH conjugates in human recombinant MPO and human liver microsomes

supplemented with an excess of the thiol nucleophile. Compound 1 was also devoid of

reversible and time-dependent inhibitory effects against major human cytochrome P450 enzymes

(Pfizer data on file), which made it an attractive candidate for advancement in preclinical toxicity

studies. In contrast with the metabolic resistance in human hepatic tissue, compound 1 was

converted to cyclic ether M1 in NADPH-supplemented rat and dog liver microsomes. Heat

inactivation which abolishes FMO activity while preserving P450 activity (Ziegler, 1980)

provided circumstantial evidence for the involvement of an FMO isoform(s) in the formation of

M1. Consistent with this initial finding, the non-selective P450 inactivator 1-aminobenzotriazole

had little effect on the conversion of 1 to M1 in rat and dog liver microsomes. In contrast, the

FMO1 competitive inhibitor imipramine dramatically reduced the oxidative desulfuration in rat


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and dog liver microsomes, respectively, implying that the conversion of 1 to M1 in rat and dog

liver microsomes is facilitated primarily by FMO1 rather than P450s. The possibility of

imipramine’s inhibitory effects occurring through inhibition of rat CYP isoforms (Murray and

Field, 1992; Masubuchi et al., 1995) can be ruled out on the basis of the results obtained with 1-

aminobenzotriazole.

Mammalian FMOs (E.C.1.14.12.8) comprise a group of flavin adenine dinucleotide-

containing enzymes that utilize NADPH and molecular oxygen to generate a 4α-

hydroperoxyflavin intermediate, which mediates the two-electron oxidation of soft, highly

polarizable nucleophilic heteroatom (nitrogen, sulfur, and phosphorus)-containing xenobiotics

(Cashman, 1995; Cashman et al., 1995, Hines et al., 1994; Krueger and Williams, 2005; Phillips

and Shephard, 2008; Ziegler, 2002). Our findings on the facile decomposition of 1 to M1 in the

presence of H2O2 and FMO1 are consistent with the notion that FMO1 will generally oxygenate

any nucleophilic heteroatom-containing compound that can be oxidized by H2O2 and/or peracids

(Bruice et al., 1983). To date, five distinct forms of FMO (i.e., FMO1–5) have been identified

(Hernandez et al., 2004; Lawton et al., 1994). Examinations of adult human liver mRNA

indicate high FMO3 (and FMO5) expression but low FMO1 expression (Dolfin et al., 1996;

Hines, 2006; Koukouritaki et al., 2002; Koukouritaki and Hines, 2005; Shimizu et al., 2011;

Zhang and Cashman, 2006; Chen et al., 2016). In contrast, rat livers have been shown to express

high levels of FMO1 protein (Cherrington et al., 1998; Itoh et al., 1993; Lattard et al., 2002a;

Yamazaki et al., 2014). Expression of FMO1 and FMO3 in dog liver has also been reported with

84–89% amino acid sequence identity to the corresponding orthologs from rat and human


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(Lattard et al., 2002b; Ripp et al., 1999; Stevens et al., 2003). As such, dog FMO1 and dog

FMO3 exhibit only 56% identities in primary amino acid sequence (Lattard et al., 2002b).

To identify the specific FMO isoform responsible for the oxidative desulfuration of 1, studies

were conducted using recombinant human FMO isoforms. FMO3 and FMO5 were used since

they represent isoforms most abundant in human liver and FMO1 because it is the ortholog of the

form most abundant in adult rat and dog liver. The formation of M1 was principally mediated by

recombinant FMO1 with little to no contribution from FMO3 or FMO5 (apparent CLint for M1

formation by FMO1 was 154-fold higher than that by FMO3), which is consistent with the

inhibitory effects of imipramine on oxidative desulfuration of 1 in rat and dog liver microsomes.

Overall, these results suggest that liver microsomal FMO1 could contribute to the relatively high

FMO-mediated oxidative desulfuration of 1 in rat and dog liver microsomes and that lower

expression of FMO1 in human livers is a major determinant of oxidation potential in livers from

preclinical species and humans. The in vitro metabolic instability of 1 also manifested in

moderate to high CLp in rats and dogs with significant circulating M1 concentrations measured

in both species, which provided an in vivo context for the in vitro findings, especially when

considering that renal excretion of 1 in unchanged form was negligible in rats and dogs.

Additional case studies on species differences in FMO-mediated metabolism have also appeared

in the literature, which strengthen our observations on the selective nature of FMO1-mediated

oxidative desulfuration in 1. For instance, a recent report from Liu et al., (2013) demonstrated

that quinuclidine ring N-oxidation in a selective α7 neuronal acetylcholine receptor agonist

ABT-107 occurred primarily in liver microsomes from rat and dog (but not in human), and was

also principally mediated by FMO1. Because mRNA expression levels for FMO1 are higher in


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human kidney (relative to liver) (Dolphin et al., 1996; Zhang and Cashman, 2006; Chen et al.,

2016), the oxidative desulfuration of 1 was also examined in NADPH-supplemented human

kidney microsomes. Compared with recombinant FMO1, 1 was relatively stable in kidney

microsomes with minimal amount of M1 (~ 80 nM) formed in a NADPH-dependent fashion.

Because of the unknown amount of FMO in the commercial preparation of the human kidney

microsomes, no rate comparisons can be made between recombinant and microsomal

preparations at the present time.

Mammalian FMOs typically display high activity toward S-oxidation in thioureas, thiones,

and thiocarbamides. The initial oxygenation of the sulfur atom produces the electrophilic

sulfenic acid (R-SOH) species that is capable of reacting with nucleophiles, including GSH

(Decker and Doerge, 1992; Henderson et al., 2004; Kim and Ziegler, 2000; Krieter et al., 1984;

Neal and Halpert, 1982; Onderwater et al., 1999; Poulsen et al., 1979; Smith and Crespi, 2002).

The sulfenic acid derivatives can undergo redox cycling in the presence of GSH coupled with the

oxidation of GSH to GSSG. The sulfenic acid metabolite can also undergo a second oxidation

by FMO to the unstable sulfinic acid (R-SO2H), which is more reactive than the sulfenic acid

metabolite and can damage the cell directly or alkylate proteins (Ji et al., 2007; Onderwater et al.,

1999; 2004). Our investigations on the oxidative desulfuration of 1 to M1 (Figure 8) largely

parallel the mechanistic insights noted in the literature. For example, addition of excess GSH

essentially eliminated the conversion of 1 to M1 in NADPH-supplemented rat and dog liver

microsomes suggesting that the initial FMO1-mediated S-oxygenation of 1 leads to the

corresponding sulfenic acid derivative 3 that undergoes redox cycling to the parent compound 1

in a GSH-dependent fashion (presumably via oxidation of GSH to GSSG). A second oxidation


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of the sulfenic acid derivative 3 yields the more electrophilic sulfinic acid species 4 that is

trapped intramolecularly (perhaps in a diffusion-controlled fashion) by the pendant alcohol motif

on the N1-substitutent in 1.

In conclusion, our studies underscore one of the limitations of rat and dog as surrogates of

adult human FMO-dependent drug metabolism studies and the conclusions from previous animal

studies that lack significant amounts of liver FMO3 (i.e., rats and dogs) may need to be

reconsidered. From a drug discovery perspective, our findings provide a cautionary note against

the use of allometric scaling of clearance from animals to human without a thorough knowledge

of the overall disposition/metabolic elimination mechanism of the molecule(s) under

consideration.


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Authorship Contributions

Participated in research design: Heather, Eng, Raman Sharma, Angela Wolford, and

Amit S. Kalgutkar

Conducted in vitro experiments: Heather Eng, Raman Sharma, and Edward L. Conn

Contributed new reagents or analytic tools: Edward L. Conn, and Roger B. Ruggeri

Performed data analysis: Heather Eng, Raman Sharma, Angela Wolford, Deepak Dalvie,

and Amit S. Kalgutkar

Wrote or contributed to the writing of the manuscript: Heather Eng, Deepak K. Dalvie,

Leonard Buckbinder, Roger B. Ruggeri, Li Di, and Amit S. Kalgutkar


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References

Boily MO, Chauret N, Laterreur J, Leblond FA, Boudreau C, Duquet MC, Lévesque JF, Ste-

Marie L, and Pichette V (2015) In vitro and in vivo mechanistic studies toward

understanding the role of 1-aminobenzotriazole in rat drug-drug interactions. Drug Metab

Dispos 43:1960-1965.

Bruice TC, Noar JB, Ball SS, and Venkataram UV (1983) Monooxygen donation potential of 4a-

hydroperoxyflavins as compared with those of percarboxylic acid and other hydroperoxides.

Monooxygen donation to olefin, tertiary amine, alkyl sulfide, and iodide ion. J Am Chem Soc

105:2452-2463.

Caldwell GW, Ritchie DM, Masucci JA, Hageman W, Cotto C, Hall J, Hasting B, and Jones W

(2005) The use of the suicide CYP450 inhibitor ABT for distinguishing absorption and

metabolism processes in in-vivo pharmacokinetic screens. Eur J Drug Metab Pharmacokinet

30:75-83.

Cashman JR (1995) Structure and catalytic properties of the mammalian flavin-containing

monooxygenase. Chem Res Toxicol 8:165-181.

Cashman JR, Park SB, Berkman CE, and Cashman LE (1995) Role of hepatic flavin-containing

monooxygenase 3 in drug and chemical metabolism in adult humans. Chem Biol Interact

96:33-46.

Chen Y, Zane NR, Thakker DR, and Wang MZ (2016) Quantification of flavin-containing

monooxygenases 1, 3 and 5 in human liver microsomes by UPLC-MRM-based targeted

quantitative proteomics and its application to the study of ontogeny. Drug Metab Dispos

pii.dmd.115.067538.


at ASPE



Dow

nloaded from


DMD # 70185

24

Cherrington NJ, Cao Y, Cherrington JW, Rose RL, and Hodgson E (1998) Physiological factors

affecting protein expression of flavin-containing monooxygenases 1, 3 and 5. Xenobiotica

28:673-682.

Cooper DS (2005) Antithyroid drugs. N Engl J Med 352:905-917.

Decker CJ and Doerge DR (1991) Rat hepatic microsomal metabolism of ethylenethiourea.

Contributions of the flavin-containing monooxygenase and cytochrome P-450 isozymes.

Chem Res Toxicol 4:482-489.

Decker CJ and Doerge DR (1992) Covalent binding of 14C- and 35S-labeled thiocarbamides in

rat hepatic microsomes. Biochem Pharmacol 43:881-888.

Dixit A and Roche TE (1984) Spectrophotometric assay of the flavin containing monooxygenase

and changes in its activity in female mouse liver with nutritional and diurnal conditions. Arch

Biochem Biophys 233:50-63.

Dolphin CT, Cullingford TE, Shephard EA, Smith RL, and Phillips IR (1996) Differential

developmental and tissue-specific regulation of expression of the genes encoding three

members of the flavin-containing monooxygenase family of man, FMO1, FMO3 and FMO4.

Eur J Biochem 235:683-689.

Futcher PH and Massie E (1950) Agranulocytosis due to propylthiouracil. Ann Intern Med

32:137.


at ASPE



Dow

nloaded from


DMD # 70185

25

Henderson MC, Krueger SK, Stevens JF, and Williams DE (2004) Human flavin-containing

monooxygenase form 2 S-oxygenation: sulfenic acid formation from thioureas and oxidation

of glutathione. Chem Res Toxicol 17:633-640.

Hernandez D, Janmohamed A, Chandan P, Phillips IR, and Shephard EA (2004) Organization

and evolution of the flavin-containing monooxygenase genes of human and mouse:

identification of novel gene and pseudogene clusters. Pharmacogenetics 14:117-130.

Hines RN, Cashman JR, Philpot RM, Williams DE, and Ziegler DM (1994) The mammalian

flavin-containing monooxygenases: molecular characterization and regulation of expression.

Toxicol Appl Pharmacol 125:1-6.

Hines RN (2006) Developmental and tissue-specific expression of human flavin-containing

monooxygenases 1 and 3. Expert Opin Drug Metab Toxicol 2:41-49.

Ichiki Y, Akahoshi M, Yamashita N, Morita C, Maruyama T, Horiuchi T, Hayashida K,

Ishibashi H, and Niho Y (1998) Propylthiouracil-induced severe hepatitis: a case report and

review of the literature. J Gastroenterol 33:747-750.

Itoh K, Kimura T, Yokoi T, Itoh S, and Kamataki T (1993) Rat liver flavin-containing

monooxygenase (FMO): cDNA cloning and expression in yeast. Biochim Biophys Acta

1173:165-171.

Ji T, Ikehata K, Koen YM, Esch SW, Williams TD, and Hanzlik RP (2007) Covalent

modification of microsomal lipids by thiobenzamide metabolites in vivo. Chem Res Toxicol

20:701-708.


at ASPE



Dow

nloaded from


DMD # 70185

26

Jiang X, Khursigara G, and Rublin RL (1994) Transformation of lupus-inducing drugs to

cytotoxic products by activated neutrophils. Science 266:810-813.

Kalm MJ (1961) Peroxide desulfurization of thioureas. J Org Chem 26:2925-2929.

Kim YM and Ziegler DM (2000) Size limits of thiocarbamides accepted as substrates by human

flavin-containing monooxygenase 1. Drug Metab Dispos 28:1003-1006.

Kitamura IR (1934) The action of hydrogen peroxide on organic sulfur compounds. Yakugaku

Zasshi 54:1-13.

Koukouritaki SB, Simpson P, Yeung CK, Rettie AE, and Hines RN (2002) Human hepatic

flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression.

Pediatr Res 51:236-243.

Koukouritaki SB and Hines RN (2005) Flavin-containing monooxygenase genetic

polymorphism: impact on chemical metabolism and drug development. Pharmacogenomics

6:807-822.

Krieter PA, Ziegler DM, Hill KE, and Burk RF (1984) Increased biliary GSSG efflux from rat

livers perfused with thiocarbamide substrates for the flavin-containing monooxygenase. Mol

Pharmacol 26:122-127.

Krueger SK and Williams DE (2005) Mammalian flavin-containing monooxygenases:

structure/function, genetic polymorphisms and role in drug metabolism. Pharmacol Ther

106:357-387.


at ASPE



Dow

nloaded from


DMD # 70185

27

Lattard V, Lachuer J, Buronfosse T, Garnier F, and Benoit E (2002a) Physiological factors

affecting the expression of FMO1 and FMO3 in the rat liver and kidney. Biochem Pharmacol

63:1453-1464.

Lattard V, Longin-Sauvageon C, Lachuer J, Delatour P, and Benoit E (2002b) Cloning,

sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1

and FMO3 in the dog. Drug Metab Dispos 30:119-128.

Lawton MP, Cashman JR, Cresteil T, Dolphin CT, Elfarra AA, Hines RN, Hodgson E, Kimura

T, Ozols J, Phillips IR et al. (1994) A nomenclature for the mammalian flavin-containing

monooxygenase gene family based on amino acid sequence identities. Arch Biochem Biophys

308:254-257.

Lee E, Hirouchi M, Hosokawa M, Sayo H, Kohno M, and Kariya K (1988) Inactivation of

peroxidases of rat bone marrow by repeated administration of propylthiouracil is

accompanied by a change in the heme structure. Biochem Pharmacol 37:2151-2153.

Lee E, Miki Y, Katsljira H, and Kariya K (1990) Mechanism of inactivation of myeloperoxidase

by propylthiouracil. Biochem Pharmacol 39:1467-1471.

Lee SK, Kang MJ, Jin C, In MK, Kim DH, and Yoo HH (2009) Flavin-containing

monooxygenase 1-catalysed N,N-dimethylamphetamine N-oxidation. Xenobiotica 39:680-

686.


at ASPE



Dow

nloaded from


DMD # 70185

28

Liu H, Deng X, Liu J, Liu N, Stuart P, Xu H, Guan Z, Marsh KC, and De Morais S (2013)

Species-dependent metabolism of a novel selective a7 neuronal acetylcholine receptor

agonist ABT-107. Xenobiotica 43:803-816.

Manivel JC, Bloomer JR, and Snover DC (1987) Progressive bile duct injury after thiabendazole

administration. Gastroenterology 93:245–249.

Martinez-Lopez JI, Greenberg SE, and Kling RR (1962) Drug-induced hepatic injury during

methimazole therapy. Gastroenterology 43:84-87.

Masubuchi Y, Takahashii C, Fujio N, Horie T, Suzuki T, Imaoka S, Funae Y, and Narimatsu S

(1995) Inhibition and induction of cytochrome P450 isozymes after repetitive administration

of imipramine in rats. Drug Metab Dispos 23:999-1003.

Mizutani T, Yoshida K, and Kawazoe S (1993) Possible role of thioformamide as a proximate

toxicant in the nephrotoxicity of thiabendazole and related thiazoles in glutathione-depleted

mice: structure-toxicity and metabolic studies. Chem Res Toxicol 6:174-179.

Mizutani T, Yoshida K, Murakami M, Shirai M, and Kawazoe S (2000) Evidence for the

involvement of N-methylthiourea, a ring cleavage metabolite, in the hepatoxicity of

methimazole in glutathione-depleted mice: structure-toxicity and metabolic studies. Chem

Res Toxicol 13:170-176.

Murray M and Field SL (1992) Inhibition and metabolite complexation of rat hepatic

microsomal cytochrome P450 by tricyclic antidepressants. Biochem Pharmacol 43:2065-

2071.


at ASPE



Dow

nloaded from


DMD # 70185

29

Neal RA and Halpert J (1982) Toxicology of thiono-sulfur compounds. Annu Rev Pharmacol

Toxicol 22:321-339.

Onderwater RC, Commandeur JN, Menge WM, and Vermeulen NP (1999) Activation of

microsomal glutathione S-transferase and inhibition of cytochrome P450 1A1 activity as a

model system for detecting protein alkylation by thiourea-containing compounds in rat liver

microsomes. Chem Res Toxicol 12:396-402.

Onderwater RC, Commandeur JN, and Vermeulen NP (2004) Comparative cytotoxicity of N-

substituted N’-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices. Toxicology

197:81-91.

Parrish KE, Mao J, Chen J, Jaochico A, Ly J, Ho Q, Mukadam S, and Wright M (2015) In vitro

and in vivo characterization of CYP inhibition by 1-aminobenzotriazole in rats. Biopharm

Drug Dispos doi: 10.1002/bdd.2000.

Phillips IR and Shephard EA (2008) Flavin-containing monooxygenases: mutations, disease and

drug response. Trends Pharmacol Sci 29:294-301.

Poulsen LL, Hyslop RM, and Ziegler DM (1979) S-Oxygenation of N-substitutedthioureas

catalyzed by the pig liver microsomal FAD-containing monooxygenase. Arch Biochem

Biophys 198:78-88.

Ripp SL, Itagaki K, Philpot RM, and Elfarra AA (1999) Species and sex differences in

expression of flavin-containing monooxygenase form 3 in liver and kidney microsomes.

Drug Metab Dispos 27:46-52.


at ASPE



Dow

nloaded from


DMD # 70185

30

Ruggeri RB, Buckbinder L, Bagley SW, Carpino PA, Conn EL, Dowling MS, Fernando DP, Jiao

W, Kung DW, Orr ST, Qi Y, Rocke BN, Smith A, Warmus JS, Zhang Y, Bowles D,

Widlicka DW, Eng H, Ryder T, Sharma R, Wolford A, Okerberg C, Walters K, Maurer TS,

Zhang Y, Bonin PD, Spath SN, Xing G, Hepworth D, Ahn K, and Kalgutkar AS (2015)

Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-

1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase

Inhibitor for the Treatment of Cardiovascular Diseases. J Med Chem 58:8513-8528.

Shimizu M, Denton T, Kozono M, Cashman JR, Leeder JS, and Yamazaki H (2011)

Developmental variations in metabolic capacity of flavin-containing monooxygenase 3 in

childhood. Br J Clin Pharmacol 71:585-591.

Smith PB and Crespi C (2002) Thiourea toxicity in mouse C3H/10T1/2 cells expressing human

flavin-dependent monooxygenase 3. Biochem Pharmacol 63:1941-1948.

Stevens JC, Melton RJ, Zaya MJ, and Engel LC (2003) Expression and characterization of

functional dog flavin-containing monooxygenase 1. Mol Pharmacol 63:271-275.

Strelevitz TJ, Foti RS, and Fisher MB (2006) In vivo use of the P450 inactivator 1-

aminobenzotriazole in the rat: varied dosing route to elucidate gut and liver contributions to

first-pass and systemic clearance. J Pharm Sci 95:1334-1341.

Tidén AK, Sjögren T, Svensson M, Bernlind A, Senthilmohan R, Auchère F, Norman H,

Markgren PO, Gustavsson S, Schmidt S, Lundquist S, Forbes LV, Magon NJ, Paton LN,

Jameson GN, Eriksson H, and Kettle AJ (2011) 2-Thioxanthines are mechanism-based


at ASPE



Dow

nloaded from


DMD # 70185

31

inactivators of myeloperoxidases that block oxidative stress during inflammation. J Biol

Chem 286:37578-37589.

Waldhauser L and Uetrecht J (1991) Oxidation of propylthiouracil to reactive metabolites by

activated neutrophils. Implications for granulocytosis. Drug Metab Dispos 19:354-359.

Yamazaki M, Shimizu M, Uno Y, and Yamazaki H (2014) Drug oxygenation activities mediated

by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats,

and minipigs. Biochem Pharmacol 90:159-165.

Zhang J and Cashman JR (2006) Quantitative analysis of FMO gene mRNA levels in human

tissues. Drug Metab Dispos 34:19-26.

Ziegler DM (1980) Microsomal flavin-containing monooxygenase: Oxygenation of nucleophilic

nitrogen and sulfur compounds. Enzymatic Basis of Detoxication (Academic Press, New

York), 1:201–227.

Ziegler DM (2002) An overview of the mechanism, substrate specificities and structure of

FMOs. Drug Metab Rev 34:503-511.


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Declaration of interest

H.E., R.S., A.W., L.D., R.B.R., L.B. E.L.C., D.D. and A.S.K. are employees of, and/or hold

stock in, Pfizer Inc.


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Figure Legends

Figure 1. Oxidative desulfuration of the N1-substituted-6-arylthiouracil 1 by MPO or H2O2.

Figure 2. HPLC-UV (λ = 280 nm) chromatogram of an incubation mixture of 1 (10 μM) in

NADPH-supplemented rat (panel A), dog (panel B), and human (panel C) liver microsomes.

Figure 3. HPLC-UV chromatogram of an incubation mixture of 1 (10 μM) in NADPH-

supplemented rat liver microsomes in the absence or presence of GSH.

Figure 4. Oxidative desulfuration of 1 (1 μM, ●) to M1 (□) in NADPH-supplemented rat liver

microsomes in the absence (panel A) or presence of heat inactivation (5 min, 50 °C) (panel B).

Symbols depict means and error bars for standard deviations.

Figure 5. Oxidative desulfuration of 1 (1 μM) to M1 in human recombinant FMO1, FMO3, and

FMO5. Incubations were conducted using 0.5 mg/mL Supersomes in the presence of NADPH

(1.3 mM) for 5 min at 37 °C. Symbols depict means and error bars for standard deviations.

Figure 6. Kinetics of oxidative desulfuration of 1 to M1 by human recombinant FMO1 (panel A,

0.1 mg/mL microsomal protein) and FMO3 (panel B, 1 mg/mL). Incubations were conducted in

the presence of NADPH (1.3 mM) at 37 °C for 60 min in triplicate. Symbols depict means and

error bars for standard deviations.


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Figure 7. Oxidative desulfuration of 1 (1 μM) to M1 in NADPH-supplemented rat (panel A) and

dog (panel B) liver microsomes in the absence or presence of FMO1 and CYP inhibitors,

imipramine (250 μM) and 1-aminobenzotriazole (1000 μM), respectively. Symbols depict means

and error bars for standard deviations.

Figure 8. Proposed mechanism of oxidative desulfuration of 1 to M1 in NADPH-supplemented

rat and dog liver microsomes.


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Tables

TABLE 1

Mean pharmacokinetic parameters of compound 1 and cyclic ether metabolite M1 in after i.v. (1

mg/kg) administration of 1 to Wistar-Han rats and Beagle dogs.

ai.v. administration of 1 in 5% DMSO/95% of 30% 2-hydroxypropyl-β-cyclodextrin. bconcentrations of M1were estimated in animals administered with 1 via the i.v. route. ci.v. administration of 1 as a solution in 25% 2-hydroxypropyl-β-cyclodextrin/ Tris buffer (100 mM) (pH = 8.0). N.A. not applicable. Pharmacokinetic parameters are expressed as mean ± standard deviation.

Compound Species Dose (mg/kg)

CLp (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

AUC(0-∞) (ng.h/mL)

1 Ratsa 1.0 (n=3)

73 ± 13 1.4 ± 0.5 0.4 ± 0.2 233 ± 37

M1b N.A. N.A. 0.8 ± 0.3 49.8 ± 11.2

1 Dogsc 1.0 (n=3)

12 ± 3.0 1.3 ± 0.3 5.3 ± 0.8 1450 ± 310

M1b N.A. N.A. 6.3 ± 0.8 769 ± 136


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Figure 2

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