Functional assessment of rat pulmonary flavin-containing monooxygenase activity

Article abstract of DOI:10.1080/00498254.2018.1469804

The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model.Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible.The predicted lung clearance (CL_lung) estimated from microsomes and slices was 16?±?0.6 and 2.1?±?0.3?mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CL_lung using rat lung microsomes corresponded to approximately 35% of rat CL_liver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine.Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

Full text of DOI:10.1080/00498254.2018.1469804

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Functional assessment of rat pulmonary flavin-
containing monooxygenase activity
Yildiz Yilmaz, Gareth Williams, Nenad Manevski, Markus Walles, Stephan
Krähenbühl & Gian Camenisch
To cite this article: Yildiz Yilmaz, Gareth Williams, Nenad Manevski, Markus Walles, Stephan
Krähenbühl & Gian Camenisch (2018): Functional assessment of rat pulmonary flavin-containing
monooxygenase activity, Xenobiotica, DOI: 10.1080/00498254.2018.1469804
To link to this article: https://doi.org/10.1080/00498254.2018.1469804
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XENOBIOTICA
https://doi.org/10.1080/00498254.2018.1469804
RESEARCH ARTICLE
Functional assessment of rat pulmonary flavin-containing
monooxygenase activity
a
a
a
a
b
a
€
€
Yildiz Yilmaz , Gareth Williams , Nenad Manevski , Markus Walles , Stephan Krahenbuhl and Gian Camenisch
b
aPharmacokinetic Sciences, Novartis Institutes for Biomedical Research, Basel, Switzerland; Clinical Pharmacology and Toxicology, University
Hospital, Basel, Switzerland
ABSTRACT
ARTICLE HISTORY
Received 21 March 2018
Revised 23 April 2018
Accepted 24 April 2018
1. The expression of flavin-containing monooxygenase (FMO) varies extensively between
human and commonly used preclinical species such as rat and mouse. The aim of this study
was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the
contribution of rat lung to the clearance of benzydamine was investigated using an in vivo
pulmonary extraction model.
KEYWORDS
Pulmonary metabolism;
flavin-containing monooxy-
genases (FMO);
2. Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal
inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is pre-
dominantly FMO mediated while any contribution from CYPs is negligible.
3. The predicted lung clearance (CLlung) estimated from microsomes and slices was 16 0.6
and 2.1 0.3 mL/min/kg, respectively. The results from in vivo pulmonary extraction indi-
cated no pulmonary extraction following intravenous and intra-arterial dosing to rats.
Interestingly, the predicted CLlung using rat lung microsomes corresponded to approxi-
mately 35% of rat CLliver suggesting that the lung makes a smaller contribution to the
whole body clearance of benzydamine.
benzydamine; rat pumonary
FMO metabolism;
benzydamine noxygenation
4. Although benzydamine clearance in rat appears to be predominantly mediated by hepatic
metabolism, the data suggest that the lung may also make a smaller contribution to its
whole body clearance.
Introduction
oxidoreductase to facilitate electron transfer from NADPH.
FMOs are not dependent on a coenzyme and substrates
undergo a one-step two-electron oxygenation. While CYPs
display optimum activity at physiological pH, FMOs display
optimum catalytic activity in the pH range from 8 to 10
(Cashman, 2005; Hutzler & Zientek, 2016).
The expression of FMOs varies markedly with species, tis-
sue, age and also gender (Hines, 2006; Phillips & Shephard,
2017; Ripp et al., 1999). For example, the major FMO isoforms
expressed in adult human liver are FMO3 and FMO5
(Cashman & Zhang, 2006; Dolphin et al., 1991; Janmohamed
et al., 2004; Koukouritaki et al., 2002). Hepatic expression of
FMO1 in human stops after birth, but it is still expressed in
extrahepatic tissues including kidney (Dolphin et al., 1996;
Zhang & Cashman, 2005). On the other hand, FMO1 and
FMO3 are both expressed in rat (Itoh et al., 1993; Lattard
et al., 2002a; Moroni et al., 1995) and mouse liver albeit in a
gender dependent manner (Cherrington et al., 1998; Falls
et al., 1995; Janmohamed et al., 2004). Such differences in
enzyme expression between preclinical species and human
The flavin-containing monooxygenases (FMOs) are a family of
enzymes that typically catalyze the oxygenation of substrates
containing a soft nucleophilic heteroatom such as nitrogen or
sulfur. Human FMO5 has recently also been shown to behave
as a Baeyer–Villiger monooxygenase, catalyzing the conver-
sion of a lactone or ketone to an ester (Fiorentini et al., 2017;
Lai et al., 2010; Meng et al., 2015). In humans, the FMO family
is relatively small and only five functional genes (FMO1–5)
have been identified (Lawton et al., 1994; Phillips et al., 1995).
FMO substrates include dietary compounds, insecticides and
drugs such as trimethylamine (Dolphin et al., 1997), phorate
and disulfoton (Henderson et al., 2004), imipramine (Furnes &
Schlenk, 2004), clozapine (Tugnait et al., 1997) and ethiona-
mide (Henderson et al., 2008).
Cytochrome P450 enzymes (CYPs) and FMOs are both
located in the microsomal compartment and are dependent
on NADPH as a cofactor (Cashman, 2005; Hutzler & Zientek,
2016). However, CYP-mediated oxidative reactions proceed
via two sequential one-electron transfer and require an may lead to significant differences in metabolism, clearance
CONTACT Yildiz Yilmaz
yildiz.yilmaz@novartis.com
Novartis Institute for Biomedical Research Pharmacokinetic Sciences, Fabrikstrasse 14-1, Novartis
Pharma AG, CH-4002 Basel, Switzerland
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y. YILMAZ ET AL.
and exposure of parent compound and metabolites, and may 2017). Although lung tissue slices are not commonly used in
alter efficacy and toxicity of drugs.
drug metabolism, they have the advantage of containing a
The liver is known to be the major site of drug metabol- variety of different cell types with differing metabolic activity.
They also retain the complex cellular architecture of the
tissue and are therefore a suitable system for the investiga-
tion of pulmonary metabolism. In humans, benzydamine
N-oxygenation has been shown to be mediated by FMO1
and FMO3 with a minor contribution from CYPs (Lang &
Rettie, 2000). In order to determine the contribution of FMOs
and CYPs to benzydamine N-oxygenation in rat lung micro-
somes, experiments using thermal inactivation and the non-
specific irreversible CYP inhibitor, 1-aminobenzotriazole (ABT)
was performed. FMOs are known to be thermally labile in
the absence of NADPH and are readily deactivated, whereas
CYP activity is reported to be relatively unaffected (Ziegler,
1980). However, FMO2 has been shown to be less labile to
heat inactivation than other isoforms such as FMO3
(Cashman, 2005; Hutzler & Zientek, 2016). This heat lability
has often been exploited to understand the relative contribu-
tions of FMOs and CYPs (Cashman, 2005; Fisher et al., 2002;
Rawden et al., 2000; Yanni et al., 2008). The in vivo clearance
of benzydamine in rat has previously been shown to be well
predicted by liver microsomes and hepatocytes, suggesting
that its elimination is mainly driven by hepatic metabolism in
this species (Fisher et al., 2002). Yet, no attempt has previ-
ously been made to determine if the lung also contributes to
the whole body clearance of benzydamine in rats. Herein,
in vitro data and the well-stirred model have been used to
predict lung clearance (CLlung). Finally, this predicted CLlung
has been compared to in vivo pulmonary extraction of ben-
zydamine following intra-arterial (i.a.; post-lungs) and intra-
venous (i.v.; pre-lungs) dosing to rats.
ism in the body. Yet, drug metabolizing enzymes such as
CYPs, uridine 50diphosphoglucuronosyltransferases (UGT) and
FMOs are also expressed in the lung (Ding & Kaminsky, 2003;
Gundert-Remy et al., 2014; Jhajra et al., 2012; Rettie et al.,
1995). The major FMO isoform expressed in the lungs of
most mammals is FMO2 (Dolphin et al., 1998; Krueger et al.,
ꢀ
2001). Caucasians and Asians possess the FMO2 2 allele that
codes for a non-functional, truncated protein. On the other
hand, 27% and 5% of African and Hispanic populations,
ꢀ
respectively, possess the FMO2 1 allele and express the full
length, functional active protein (Dolphin et al., 1998;
Krueger et al., 2004; Whetstine et al., 2000). Similarly, the
FMO2 gene in commonly used laboratory rat strains has also
been shown to encode a truncated, inactive protein (Lattard
et al., 2002b), whereas full-length functional FMO2 is
expressed in mouse lung (Karoly and Rose, 2001). FMO1 has
also been shown to be expressed in both mouse and rat
lung (Janmohamed et al., 2004; Lattard et al., 2001). Despite
these species differences in pulmonary expression, it can be
anticipated that in rats and mice, FMO substrates will be sub-
ject to lung metabolism. However, pulmonary metabolism of
FMO substrates in humans will depend upon the popula-
tion involved.
Metabolism in organs such as the lung may also contrib-
ute to systemic clearance and elimination of compounds.
Given that the lung is a well-perfused organ that receives a
blood flow equivalent to cardiac output (Davies & Morris,
1993), it is reasonable to assume that it may play a role in
extrahepatic metabolism. Indeed, in vivo pulmonary metabol-
ism has been demonstrated in rodents for lidocaine, midazo-
lam and nifedipine (Aoki et al., 2010), resveratrol (Sharan &
Materials and methods
Nagar, 2013) and phenol (Cassidy
& Houston, 1980).
Reagents, chemicals and materials
Although FMOs are known to be expressed in the lungs of
rodents, the contribution of pulmonary metabolism to the
clearance of FMO substrates in vivo is yet to be explored.
The aim of this investigation was to evaluate pulmonary
FMO activity in rats using the anti-inflammatory drug, ben-
zydamine, a well characterized, high-turnover FMO substrate
Benzydamine hydrochloride, benzydamine N-oxide maleate,
NADPH, sulfadimethoxine (SDM) and 1-aminobenzotriazole
were purchased from Sigma-Aldrich (Poole, UK). HPLC grade
water and acetonitrile were purchased from VWR
(Leicestershire, UK). Sprague-Dawley rat (pool of 299) lung
microsomes were obtained from Xenotech (Kansas City, MO).
Lung microsomes were not further characterized with regard
to CYP and FMO protein content.
€
(Capolongo et al., 2010; Lang & Rettie, 2000; Stormer et al.,
2000; Taniguchi-Takizawa et al., 2015). FMO catalyzes the N-
€
oxygenation of benzydamine as shown in Figure 1 (Stormer
et al., 2000). In this study, the enzyme kinetics of benzyd-
amine N-oxygenation in rat lung microsomes and rat lung sli-
ces were compared. The precision cut lung slices incubation
system described in this investigation has previously been
used to investigate pulmonary CYP1A1 activity (Yilmaz et al.,
Benzydamine N-oxygenation enzyme kinetics in rat
lung microsomes
Rat lung microsomal incubations (0.3 mL total volume) were
conducted at 37 ꢁC in 100 mM potassium phosphate buffer,
pH 7.4, supplemented with 5 mM MgCl2 and 1 mM NADPH.
FMOs are known to display optimum catalytic activity within
the pH range from 8 to 10 (Cashman, 2005; Hutzler &
Zientek, 2016) and the impact of higher pH on benzydamine
N-oxygenation has previously been demonstrated (Kawaji
et al., 1993). However, for the purposes of this investigation,
we decided that it was more appropriate to perform the
incubations at physiological pH (7.4), which reflects the
Figure 1. Benzydamine N-oxygenation and N-demethylation. For a full over-
view of the biotransformation pathway of benzydamine, refer to (Fisher
et al., 2002).

XENOBIOTICA
3
in vivo situation. For all experiments, microsomes were prein- equation.
cubated for 5 min with NADPH and reactions were started by
addition of benzydamine. Initial studies were performed to
establish conditions for linear product (benzydamine N-oxide)
Cbuffer
Chomoganate
fu;hD
¼
(2)
fu,hD was then scaled to undiluted lung homogenate (fu,lung
)
formation with respect to incubation time and microsomal
protein concentration. In these experiments, benzydamine
(1–200 mM) was incubated with rat lung (0.05 mg/mL micro-
somal protein) microsomes for 10 min. Benzydamine stock
solutions were prepared in methanol/water (1:1, v/v) at
appropriate concentrations and the concentration of organic
solvent in incubations was <0.5%. Reactions were terminated
by addition of ice-cold acetonitrile containing internal stand-
ard, SDM. Samples were centrifuged and the supernatants
were analyzed by liquid chromatography-tandem mass spec-
trometry (LC-MS/MS).
using the below equation.
1
1
ꢀ
ꢁ
fu;lung
¼
(3)
1 þ D
ꢃ 1
fu;hD
where D is the dilution factor that was used.
Prediction of pulmonary clearance
The pulmonary microsomal CLint,in-vitro (Vmax/Km) value,
332 14 mL/min/mg determined in the enzyme kinetics
experiment, was used to calculate CLint,lung as described in
Equation (4).
CLint,in-vitro obtained from the lung slices experiment was
calculated using the rate of benzydamine N-oxide formation
as represented by the slope (Njuguna et al., 2016). Lung
Enzyme kinetic parameters were calculated by the
Michaelis–Menten equation:
½ ꢂ
Vmax
S
½ ꢂ
v ¼
(1)
Km þ S
where v and Vmax are the reaction and limiting reaction
velocities, respectively, Km is the Michaelis–Menten constant
and [S] is the total substrate concentration. Prism 7.0 organ clearance (CLint,lung) was calculated using Equations (5)
(GraphPad Software, San Diego, CA) was used for data proc-
essing and the determination of kinetic parameters.
and (6):
Calculation of CLint,lung using lung microsomal data:
CLint;in vitro ꢄ ðmicrosomal content per g tissueÞ
ꢄðg tissue per kg body weightÞ
¼
fuinc
Benzydamine N-oxygenation in rat lung slices
CLint;lung
Rat lung slice preparation and rat lung slicing incubations
were performed as previously described (Yilmaz et al., 2017).
Benzydamine (1 mM) was incubated with lung tissue slices for
30 min to 4 h. The incubation volume was 2 mL. Negative
control incubations without tissue slices were performed in
parallel. Incubations were stopped with 3 mL of ice-cold
acetonitrile containing internal standard. The samples were
thoroughly mixed and transferred to 5 mL Eppendorf tubes.
The samples were stored at ꢃ80 ꢁC until further analysis.
(4)
Calculation of CLint,in-vitro and CLint,lung using lung tissue
slice data:
ðNꢃOxygenation formation rateÞ
ꢄðlL incubation volumeÞ
CLint;
¼
(5)
inꢃvitro
ð
Þ
mean substrate conc:
ꢄ ðmg lung tissue per sampleÞ
ꢂ
ꢃ
g lung tissue weight
per kg body weight
ðCLint;in vitroÞ ꢄ
CLint;lung
¼
(6)
fulung
Benzydamine lung tissue binding
CLlung values were then calculated using the well-stirred
organ model as shown in the below:
Rapid equilibrium dialysis (RED) was used to investigate the
binding of benzydamine to rat lung tissue. Tissue homogen-
ate was prepared from rat lungs that had been exsangui-
nated and perfused by cardiac puncture with 10 mL
Dulbecco’s Phosphate-Buffered Saline (DPBS) to avoid the
plasma binding bias. Rat lung homogenate (0.9 g tissue in
2 mL of DPBS) was diluted with 2 mL of DPBS. Three hundred
micro liter of tissue homogenate containing 5 mM benzyd-
amine and 500 mL of 100 mM of phosphate buffer (pH 7.4)
were added to the homogenate and buffer chambers of the
RED devices, respectively. The chambers were covered with a
gas permeable membrane and incubated for 4 h at 37 ꢁC
with 5% of CO2 on a shaker (750 rpm). Experiments were per-
formed in triplicate. Equal volumes of homogenate and buf-
fer were then taken from the chambers and transferred to a
96-deep-well plate for LC-MS/MS analysis.
QlungꢄClint;lung ꢄ fu=Rb=p
QlungþClint;lung ꢄ fu=Rb=p
CLlung
¼
(7)
where fuinc is the fraction unbound in microsomal incuba-
tions, fulung is the fraction unbound in lung tissue, fu is the
plasma protein binding and Rb/p is the blood to plasma ratio.
The following scaling factors derived from the literature
were used to calculate lung CLlung: microsomal protein in
lung 15 mg/g lung (Schmitz et al., 2008), rat weight 250 g, rat
lung weight 1.5 g and pulmonary blood flow (Qlung) 74 mL/
min/rat (Davies & Morris, 1993). In the absence of rat experi-
mental data, human fuinc, fu and Rb/p values of 0.35, 0.148
and 0.76 were used (Jones et al., 2017). The fulung for rat
lung homogenate was measured as 0.03. The mean linear
coefficient for benzydamine N-oxide formation rate in lung
The unbound fraction of benzydamine in diluted lung
homogenate (fu,hD) was calculated as shown in the below tissue slices was 0.003 0.0004 mM/min (n ¼ 3).

4
Y. YILMAZ ET AL.
acetonitrile containing SDM. Samples were stored at ꢃ80 ꢁC
until the day of analysis. Calibration curves for benzydamine
and benzydamine N-oxide were prepared in control rat
blood. The samples were thoroughly mixed, centrifuged
(3500 rpm, 10 min, 20 ꢁC) and the supernatant transferred to
a 96-plate containing an equal volume of water. Samples
were analyzed by LC-MS/MS as described below. The N-oxide
was stable under the conditions used here and there was no
indication of significant reduction during sample preparation
and analysis.
Thermal inactivation/chemical inhibition
Heat inactivation and the non-specific CYP inhibitor, ABT was
used to investigate the contribution of CYP and FMO to ben-
zydamine N-oxygenation in rat lung microsomes (Cassidy &
Houston, 1980; Fisher et al., 2002; Taniguchi-Takizawa et al.,
2015). In the control experiment, lung microsomal incuba-
tions containing 0.05 mg/mL of microsomal protein were pre-
incubated with NADPH (1 mM), for 10 min at 37 ꢁC. To
investigate the effect of CYP inhibition, microsomal mixtures
were preincubated with 1 mM of NADPH and 2 mM of ABT,
which is a pan-CYP inhibitor for 10 min prior to starting the
reactions. To determine the impact of thermal inactivation,
microsomes were incubated at 45 ꢁC for 5 min in the absence
of NADPH. These preparations were then cooled on ice for
10 min prior to being preincubated with NADPH for 10 min
at 37 ꢁC. Finally, to assess the combined effect of thermal
inactivation and CYP inhibition, heat-inactivated microsomes
were preincubated with NADPH and ABT (2 mM) for 10 min
at 37 ꢁC. In all experiments, reactions were started by the
addition of benzydamine (1 mM) and terminated at 10 min by
the addition of ice-cold acetonitrile containing internal stand-
ard, SDM. It has been indicated previously that the N-oxide is
stable under similar conditions to those used here and that
autoxidation was negligible in liver microsomal experiments
where comparable conditions were used (Yamazaki et al.,
2014). Similarly, there was no indication of autoxidation in
the lung incubations described in the current investigation.
For qualitative analysis of benzydamine metabolite pat-
terns in rat lung microsomes, incubations were performed as
described for the control incubation above but using higher
benzydamine and microsomal protein concentrations of 5 mM
and 0.25 mg/mL, respectively. Once again, reactions were
started by the addition of benzydamine and terminated at
10 min by the addition of ice-cold acetonitrile.
Non-compartmental pharmacokinetic analysis
Benzydamine and benzydamine N-oxide pharmacokinetic
parameters were calculated by non-compartmental analysis
(NCA) using Phoenix WinNonlin (Version 6.4, Pharsight
Corporation, Mountain View, CA). The trapezoidal rule was
used to calculate the area under the blood concentration–
time curve (AUC) which was extrapolated to infinity (AUC0-inf
using the apparent terminal disposition rate constant (kz).
Blood clearance (CLblood values were calculated as
)
)
CLblood ¼ dose/AUC0-inf and the half-life was estimated from
the terminal rate constant using half-life ¼ 0.693/kz. The
mean residence time (MRT, defined as the area under the
first moment curve (AUMC0-inf) divided by AUC0-inf was used
to calculate the steady-state volume of distribution (Vss)
using Vss ¼ MRT ꢄ CLblood
.
Quantitative LC-MS/MS
Quantitative analysis was performed on an LC-MS/MS system
consisting of a Waters Acquity UPLC coupled to a Quattro
Premier (Waters Corporation, Milford, MA) triple quadrupole
mass spectrometer with an electrospray ionization (ESI)
source
operated
in
positive
ionization
mode.
Chromatography was performed using a Waters Acquity
UPLC BEH C18 column (2.1 mm ꢄ 500 mm, 1.7 mm (Waters
Corporation) maintained at 45 ꢁC with a flow rate of 0.5 mL/
min. The mobile phase consisted of solvent A (0.1% of formic
acid in water) and solvent B (acetonitrile). The chromato-
graphic gradient was optimized to provide baseline separ-
ation between benzydamine and benzydamine N-oxide.
Gradient elution was as follows: 0 min ! 5% B; 0.8 min !
5% B; 3.3 min ! 50% B; 3.8 min ! 50% B; 7.4 min ! 5% B.
Analytes were detected using multiple reaction monitoring
(MRM) mode and the following transitions, cone voltage (CV)
and collision energy (CE) settings: benzydamine (m/z 310 !
86; CV, 30 V; CE, 18 eV), benzydamine-N-oxide (m/z 326 !
102; CV, 30 V; CE, 15 eV) and the internal standard, SDM (m/z
311 ! 156; CV, 30 V; CE, 30 eV). Desolvation, cone (both
nitrogen) and collision (argon) gases were set at 750 L/h,
50 L/h 2.5 ꢄ 10 ꢃ 3 mbar, respectively. The source and desol-
vation temperatures were 120 and 300 ꢁC, respectively.
Five micro liter of sample was injected into the LC-MS/
MS system.
Estimation of in vivo pulmonary extraction ratios in rat
Studies were conducted using male Sprague-Dawley rats
aged 8–9 weeks (300–350 g) supplied by Charles River Labs,
Margate, Kent. Rat pharmacokinetic studies were conducted
by the UK Metabolism and Pharmacokinetics group.
Experimental procedures were performed according to the
animal welfare regulations effective in the UK (Scientific
Procedures Act, 1986).
In vivo experiments to estimate the pulmonary first pass
extraction of benzydamine in rat were performed as
described previously (Yilmaz et al., 2017). Briefly, under anes-
thesia, the carotid artery and vena cava of the rats (n ¼ 3)
were cannulated for intra-arterial (i.a.) or intravenous (i.v.)
administration, respectively. A temporary in-dwelling tail vein
cannula was inserted into the tail vein of each rat for serial
blood sampling. A 1 mg/kg bolus dose of benzydamine dis-
solved in 5% of dextrose was administered via the carotid
artery or vena cava cannulas. Serial blood samples were then
taken at 3, 6, 15, 45, 90, 150 and 240 min post-administration
and collected into vials containing EDTA and 50 ml of the
MassLynx software (Waters Corporation) was used for the
samples were then transferred to vials and protein precipita- analysis of chromatograms. The processing of MS data was
tion immediately carried out by addition of 150 mL performed using QuanLynx software (Waters Corporation).

XENOBIOTICA
5
Enzyme kinetics of benzydamine N-oxygenation in rat liver
microsomes has been previously determined and the values
are also shown in Table 1 (Taniguchi-Takizawa et al., 2015).
Although hepatic Km and Vmax values were higher than those
obtained with lung microsomes, CLint,in-vitro (Vmax/Km) values
were reasonably comparable (280 and 332 mL/min/mg for the
rat liver and lung microsomes, respectively).
Qualitative LC-MS/MS
Qualitative analysis was performed using a Thermo LTQ
Orbitrap XL hybrid mass spectrometer (Thermo Finnigan, UK)
interfaced with an Accela HPLC system (Thermo Finnigan).
Mass spectral data were acquired in positive ESI mode with
full-scan MS spectra obtained from m/z 100 to 1300 with a
mass resolution of 30,000. Collision induced dissociation (CID)
was performed at a normalized CE of 35. Accurate measure-
ment of m/z values and data processing of extracted ion chro- Benzydamine N-oxygenation in rat lung slices
matograms (EICs) were carried out using Xcalibur 2.1 (Thermo
Benzydamine N-oxide formation was linear during the 4 h
Fisher Scientific, Waltham, MA, USA). Chromatographic separ-
incubation and terminal concentrations corresponded to
ation was achieved on an Acquity BEH C18 column
approximately 61% of the initial benzydamine concentration
(150 ꢄ 2.1 mm i.d., 1.7 mm, Waters Corporation). The reversed-
(Figure 3). The rate of benzydamine N-oxide formation in
phase gradient LC-MS experiments were performed using a
lung slices (represented by the slope, in units of concentra-
mobile phase A of 0.1% of formic acid (aqueous) and a mobile
tion/time) was used to calculate CLint,in-vitro (Njuguna et al.,
phase B of acetonitrile at a flow rate of 0.3 mL/min. The HPLC
2016). The in vitro intrinsic clearance, CLint,
was
in vitro
gradient started with 5% B (0–1 min) and linearly increased to
90% B in 18 min, followed by a decrease to 40% B in 5 min
prior to column re-equilibration.
0.069 0.01 mL/min/mg slice, resulting in an up-scaled in vitro
lung clearance CLint,lung of 14.0 2.0 mL/min/kg. The well-
stirred model was then used to calculate in vivo whole lung
clearance CLlung (2.1 0.3 mL/min/kg, Table 2).
Results
Benzydamine N-oxygenation enzyme kinetics in rat
Thermal inactivation/CYP inhibition
lung microsomes
The in vitro data obtained using heat inactivation and the
non-specific CYP inhibitor, ABT is summarized in Figure 4.
Pulmonary benzydamine N-oxide formation in incubations
containing ABT was comparable to that observed in the con-
trol incubation and there was no indication inhibition.
Conversely, heat treatment resulted in a significant reduction
Linear formation of benzydamine N-oxide was observed dur-
ing the 10 min incubation period. Pulmonary benzydamine
N-oxygenation appeared to be well described by the
Michaelis–Menten model and the calculated kinetic parame-
ters (Vmax, Km) are summarized in Figure 2 and Table 1.
Figure 3. Incubation of benzydamine in rat lung slices. Dissapearance of ben-
zydamine (circles) and formation of benzydamine N-oxide (triangles) during
the 4 h incubation with rat lung slices. Results are shown as mean
values S.D. (n ¼ 3).
Figure 2. Kinetic parameters for benzydamine N-oxygenation by rat lung
microsomes. Enzyme kinetics of benzydamine N-oxygenation by rat lung micro-
somes. Enzyme kinetic data were fitted to the Michaelis–Menten equation and
are presented as mean values S.D. (n ¼ 3).
Table 1. Kinetic parameters for benzydamine N-oxygenation mediated by rat liver and lung microsomes.
Km (mM)
Vmax (nmol/min/mg)
Vmax/Km (mL/min/mg)
Rat liver (Taniguchi-Takizawa et al., 2015)
Rat lung
22
2.9 0.2
6.1
1.0 0.03
280
332 14
The rate of benzydamine N-oxide formation was measured following incubation of benzyadamine (1–1000 mM) with rat liver
and (1–200 mM) lung microsomes (0.050 mg/mL microsomal protein) for 30 and 10 min, respectively. The values calculated in
rat liver microsomes for benzydamine were obtained from a previous study (Taniguchi-Takizawa et al., 2015). Data were fitted
according to the Michaelis–Menten equation. The estimated Km, Vmax and Vmax/Km (CLint,in-vitro) values are expressed as mean
values S.D. (n ¼ 3).

6
Y. YILMAZ ET AL.
Table 2. Prediction of rat CLorgan for benzydamine.
the CYP-mediated metabolite, nor-benzydamine, were
observed. The partial biotransformation pathway and
selected ion chromatogram are shown in Figures 1 and 5,
respectively. A more detailed investigation of the biotrans-
formation of benzydamine in rat and human has previously
been performed (Fisher et al., 2002).
Organ
CLint,organ (mL/min/kg)
CLorgan (mL/min/kg)
Liver (microsomal)
Lung (microsomal)
Lung (slices)
1365 175
85 3.5
13.9 2.0
46 1.1
16 0.6
2.1 0.3
Benzydamine CLorgan (CLlung and CLliver) values were predicted using the pul-
monary and hepatic Vmax/Km (CLint,in-vitro) values determined in the enzyme
kinetics experiments. Previously determined benzydamine N-oxygenation val-
ues obtained with rat liver microsomes are also shown. Scaling factors were
used as follows: microsomal protein in lung 15 mg/g (Schmitz et al., 2008) and
liver 45 mg/g (Obach, 2001), rat body weight 250 g, rat lung weight 1.5 g, rat
liver weight 10 g, pulmonary blood flow (Qlung) 296 mL/min/kg rat and hepatic
blood flow (Qliver) 55.2 mL/min/kg (Davies and Morris, 1993). In the absence of
rat experimental data, human fuinc, fulung (internal rat data), fu and Rb/p values
of 0.35, 0.03, 0.148 and 0.76 were used (Jones et al., 2017). Results are pre-
sented as mean values S.D. (n ¼ 3).
Estimation of in vivo pulmonary extraction in rat
Benzydamine and benzydamine N-oxide blood concentra-
tions were quantified following i.v. and i.a. administration of
benzydamine to Sprague-Dawley rats and the pharmacoki-
netic parameters and concentration-time profiles are shown
in Figure 6 and Table 3, respectively. Benzydamine was rap-
idly eliminated following i.v. and i.a. with blood clearances
estimated to be 173 and 182 mL/min/kg, respectively
(approximately three-fold higher than hepatic blood flow).
The data showed that the benzydamine exposures achieved
via the two administration routes were comparable and that
there was no indication of significant pulmonary extraction
under the conditions described here. The mean benzydamine
N-oxide exposure obtained following i.v. dosing was slightly
higher than after i.a. dosing (72 4 and 61 7 h ꢄ ng/mL
respectively). However, this may likely be due to analytical or
inter-animal variability rather than a reflection of pulmon-
ary extraction.
Discussion
Figure 4. Effect of heat treatment and ABT-mediated CYP inhibition on benzyd-
amine N-oxygenation in rat lung microsomes. The effect of thermal inactivation
and the non-specific CYP inhibitor, ABT on benzydamine N-oxygenation by rat
lung microsomes. Results are shown as mean values S.D. (n ¼ 3). N.D. indi-
cates that the N-oxide metabolite was not detected.
Preclinical species such as rats and mice are commonly used
for the optimization of pharmacokinetic properties and for
testing the in vivo efficacy of new compounds. The pharma-
cokinetic data from these preclinical species are often used
for the prediction of human pharmacokinetics. Therefore, it is
desirable that drug metabolism in these species is represen-
tative of that in human. However, the complex species
dependent expression displayed by FMOs (Hines, 2006) may
result in marked differences in metabolism and pharmacokin-
etics in preclinical species and human. For example, the
absence of FMO1 in adult human liver but expression in rat
(Itoh et al., 1993) and mouse liver (Cherrington et al., 1998;
Janmohamed et al., 2004) means that these species may not
be entirely representative for humans concerning FMO1 sub-
strates. Similarly, the pulmonary expression of functional
FMO2 in preclinical species and humans is complex. The full-
length functional protein is expressed in mouse lung (Karoly
& Rose, 2001), but not in the lungs of commonly used
laboratory rat strains such as Sprague-Dawley and Wistar
(Lattard et al., 2002b). Furthermore, 27% and 5% of people of
African descent and Hispanic populations, respectively, pos-
Figure 5. Metabolism of benzyadamine in rat lung microsomes. Selected ion
chromatogram showing benzydamine and oxidative metabolites obtained fol-
lowing incubation of benzydamine with rat lung microsomes. Selected masses
include 310.191 (benzydamine), 326.186 and 296.176 (nor-benzydamine).
ꢀ
sess the FMO2 1 allele that encodes for the full-length, func-
tional, FMO2 protein whereas in Caucasians and Asians,
of benzydamine N-oxide formation in lung fractions.
Conversely, heat treatment resulted the complete abolish- FMO2 is absent (Dolphin et al., 1998; Krueger et al., 2004;
ment of benzydamine N-oxide formation in lung fractions. Whetstine et al., 2000).
There was no indication of autoxidation in the lung incuba-
tions described in the current investigation.
The impact of this variability in pulmonary expression of
functional FMO2 in humans has been demonstrated using
The major metabolite detected in lung microsomal incu- the antibiotic, ethionamide (Henderson et al., 2008).
bations was benzydamine N-oxide while only low levels of Ethionamide has been shown to undergo significantly higher

XENOBIOTICA
7
Figure 6. Blood concentration-time profiles of benzydamine and its metabolite benzydamine N-oxide after intra-arterial and intravenous dosing. Mean blood con-
centration-time profiles for benzydamine (A) following i.v. (circles) and i.a. (squares) administration and its metabolite benzydamine N-oxide (B) after i.v. (diamonds)
and i.a. (triangles) administration to anesthetized male Sprague-Dawley rats. Results are shown as mean values S.D. (n ¼ 3).
Table 3. Pharmacokinetics of benzydamine in rat.
Benzydamine, 1 mg/kg i.a.
Benzydamine, 1 mg/kg i.v.
Analyte
Units
(n ¼ 3)
(n ¼ 3)
Benzydamine
AUCinf
CLblood
Vss
h ꢄ ng/mL
mL/min/kg
L/kg
93 15
182 31
11 2.4
0.9 0.1
98 15
173 28
12 3.1
1.1 0.2
T1/2
h
Benzydamine N-oxide
AUCinf
h ꢄ ng/mL
61
7
72 4
Measured blood concentration profiles over time of benzydamine after single i.v. and i.a. dosing to male Sprague-Dawley rats
were analyzed by non-compartmental pharmacokinetic analysis. Data are presented as mean values S.D. (n ¼ 3).
FMO2-mediated S-oxygenation in human lung microsomes the tissue slice during the incubation (de Graaf et al., 2006;
obtained from individuals expressing the functional protein Ekins et al., 1995; Worboys et al., 1997). The lung is a highly
than in lung microsomes where the truncated protein was complex organ consisting more than 40 different types of
expressed (Henderson et al., 2008). The authors also sug- cells. Therefore, a further cause of underprediction may be
gested that the expression of functional FMO2 in African and the limited number of cells in the slices that contain drug
Hispanic populations may have important implications with metabolizing enzymes (Carlile et al., 1999). Although CLlung
respect to efficacy and toxicity. For instance, an FMO2 sub- predicted with tissue slices is lower than the value obtained
strate that is intended for inhaled delivery may undergo with microsomes (2.1 0.3 and 16 0.6 mL/min/kg, respect-
metabolism in the lungs of these individuals, potentially lead- ively) both methods suggest that the contribution of the rat
ing to inactivation and therefore reduced efficacy. lung to the whole body clearance of benzydamine is negli-
Alternatively, it may lead to the formation of a metabolite gible (less than 5% pulmonary blood flow).
that causes local pulmonary toxicity.
The significant rat pulmonary activity observed in lung
The aim of this study was to investigate pulmonary FMO microsomes experiments raised the question as to whether
activity in rats using the anti-inflammatory drug, benzyd- this N-oxygenation was catalyzed by FMOs, CYPs or a com-
amine. Furthermore, in vivo experiments were performed to bination of both. FMOs are known to be thermally labile
understand if the rat lung makes a contribution to the whole (Ziegler, 1980) and therefore, care should be taken when per-
body clearance of benzydamine. Benzydamine N-oxygenation forming in vitro incubations to avoid loss of activity. As
was efficiently catalyzed by rat lung microsomes and lung sli- shown in Figure 4, heat treatment of lung microsomes at
ces (Figures 2 and 3, Tables 1 and 2). While calculated lung 45 ꢁC for 5 min completely abolished benzydamine N-oxygen-
Km and Vmax values were lower than previously determined ation in rat lung. Conversely, incubation with only ABT
liver values (Taniguchi-Takizawa et al., 2015; Yamazaki et al., appeared to have no significant impact (Figure 4). As shown
2014), CLint,in-vitro (Vmax/Km) values were reasonably compar- previously (Taniguchi-Takizawa et al., 2015), heat treatment
able (280 and 332 mL/min/mg for the rat liver and lung of rat liver microsomes in the absence of NADPH results in a
microsomes, Table 1).
marked but incomplete reduction of benzydamine N-oxygen-
CLorgan predicted from lung slices was approximately ation. The data obtained in this study suggest that in
eight-fold lower than the value calculated with lung micro- rat, pulmonary benzydamine N-oxygenation is predominantly
somes (2.1 and 16 mL/min/kg, respectively, Table 2). One of catalyzed by FMO while the contribution from CYPs
the potential drawbacks of the use of tissue slices is that this is negligible.
technique often underestimates metabolic clearance. This
The full-scan MS data obtained from the analysis of rat
underprediction has previously been attributed to poor tissue lung microsomal samples are shown in Figure 5. The metab-
penetration, diffusion and distribution of the drugs within olite pattern in rat lung was relatively simple and only

8
Y. YILMAZ ET AL.
benzydamine N-oxide and low levels of nor-benzydamine estimated from in vitro data was 46 1.1 mL/min/kg, equiva-
were detected. The formation of nor-benzydamine has been
shown to be catalyzed by CYP (Taniguchi-Takizawa et al.,
2015) and the limited formation of nor-benzydamine
observed here is consistent with the predominant assump-
tion that the expression of CYPs in lung is generally low.
From these in vitro data, we concluded that, in rat, pul-
monary benzydamine N-oxygenation is mainly catalyzed by
FMO with no significant contribution of CYPs. It should be
noted that although the involvement of FMO has been con-
firmed here, no attempt has been made to identify the spe-
cific isoforms responsible for the pulmonary N-oxygenation
of benzydamine. FMO1 is known to be expressed in rat liver
(Itoh et al., 1993) and lung (Lattard et al., 2001) and there-
fore, we may assume that this isoform contributes to the
observed pulmonary N-oxygenation. FMO3 expression in rat
lung has been shown to be relatively low and as a result,
may not play a large role in the activity observed here
(Lattard et al., 2001). While pulmonary FMO is commonly
associated with the oxygenation of sulfur containing com-
pounds, benzydamine has been shown to undergo complete
N-oxygenation following incubation for 16 h with Escherichia
coli expressing the full-length functional FMO2 protein (Geier
et al., 2015). Although it has been reported that functional
FMO2 is not expressed in the lungs of commonly used
laboratory strains (Lattard et al., 2002b), we cannot rule out
its potential involvement in the N-oxygenation observed
here. However, FMO2 is known to be less vulnerable to heat
inactivation than other isoforms (Cashman, 2005; Hutzler &
Zientek, 2016) and the complete abolishment of N-oxygen-
ation in heat treated lung microsomes observed in this study
suggests that FMO2 was not involved. However, this would
have to be confirmed with additional experiments.
lent to approximately 83% hepatic blood flow (Table 2). This
suggests that, in rat, benzydamine clearance is predominantly
mediated by hepatic metabolism. The predicted CLlung using
rat lung microsomes corresponded to approximately 35% of
rat CLliver, also suggesting that the lung makes only a small
contribution to the whole body clearance of benzydamine.
Despite these findings, there may still be value in investigat-
ing the pulmonary metabolism of further FMO substrates.
In summary, benzydamine was used as a general FMO
substrate to observe pulmonary FMO activity in rat and to
determine the contribution of the rat lung to the whole
body clearance of this compound. Although the data indicate
that clearance of benzydamine in rats is mainly due to hep-
atic metabolism, the in vitro results also suggest that the
lung makes a small contribution to systemic clearance of
benzydamine. However, this failed to translate into a measur-
able pulmonary extraction in vivo (Figure 6), at least under
the conditions used here. The route of administration may
also be an important factor to consider and it is possible that
a compound administered via inhalation may be subject to
greater first pass lung extraction than a drug administered
orally or intravenously. However, this is speculative and
would need to be confirmed via appropriate experiments.
Acknowledgements
The authors would like to acknowledge Jon Rose, Arnold Demailly,
Gaelle Chenal, Hazel Smulders and Asadh Miah for their support in the
generation of data.
Disclosure statement
Similarly to the liver, in vitro pulmonary intrinsic clearance
values and the well-stirred model may be used to predict in
vivo lung clearance. However, the well-stirred model depends
on the assumption that the cells and their metabolic activ-
ities are homogeneously distributed in the system, which
may not be the case for the lung (with 40 different cell
types). Furthermore, appropriate scaling factors are not as
well established as those used for the liver and further work
is certainly required in this area. The validity of such predic-
tions can be tested using rodent in vivo pulmonary extraction
models where the compound of interest is dosed both pre-
(i.v.) and post-lung (i.a.). This approach has been previously
used to successfully demonstrate the impact of CYP, UGT
and sulfotransferase-mediated lung metabolism (Aoki et al.,
2010; Cassidy & Houston, 1980; Sharan & Nagar, 2013).
CLlung estimated using rat lung microsomes was low
(16 0.6 mL/min/kg; Table 2) and equated to approximately
5% of pulmonary blood flow. The detection of such a low
first pass extraction in vivo may be confounded by analytical
and inter-animal variability and as such, the in vivo data
obtained following i.a and i.v. dosing failed to show a signifi-
cant difference in benzydamine exposure (Figure 6 and Table
3). Although the mean benzydamine N-oxide exposure
observed after i.v. dosing was slightly higher than after i.a.
dosing, the variability in the data preclude a confident attri-
bution to pulmonary metabolism. The predicted CLliver
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Pharmacokinetic Sciences group at
Novartis Institutes for Biomedical Research, Basel, Switzerland and
Horsham, UK.
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