New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone

Article abstract of DOI:10.1080/00498254.2017.1342288

1.Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2.We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography–tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3.A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4.The functional activity of Oxy-EK was investigated on the muscarinic receptors (M_1-3) demonstrating its lack of antimuscarinic activity. 5.Despite the presence of the α,β-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Full text of DOI:10.1080/00498254.2017.1342288

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
New insights in the metabolism of Oxybutynin:
evidence of N-oxidation of propargylamine moiety
and rearrangement to enaminoketone.
Silvio Aprile, Rossana Canavesi, Rosanna Matucci, Cristina Bellucci, Erika Del
Grosso & Giorgio Grosa
To cite this article: Silvio Aprile, Rossana Canavesi, Rosanna Matucci, Cristina Bellucci, Erika
Del Grosso & Giorgio Grosa (2017): New insights in the metabolism of Oxybutynin: evidence of
N-oxidation of propargylamine moiety and rearrangement to enaminoketone., Xenobiotica, DOI:
10.1080/00498254.2017.1342288
To link to this article: http://dx.doi.org/10.1080/00498254.2017.1342288
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Date: 15 June 2017, At: 00:57

New insights in the metabolism of Oxybutynin: evidence of N-oxidation of propargylamine
moiety and rearrangement to enaminoketone.
Silvio Aprile1*, Rossana Canavesi1, Rosanna Matucci2, Cristina Bellucci2, Erika Del Grosso1, and
Giorgio Grosa1
1Department of Pharmaceutical Sciences – Laboratory of pharmaceutical analysis, University of
Eastern Piedmont, L.go Donegani 2, 28100 Novara, Italy. 2Department of Neuroscience,
Psychology, Drug Research and Child’s Health, University of Florence, V.le G. Pieraccini 6, 50139
Firenze, Italy.
*Corresponding author: Dr. Silvio Aprile; Department of Pharmaceutical Sciences – Laboratory
of pharmaceutical analysis, University of Eastern Piedmont. L.go Donegani 2, 28100 Novara, Italy.
tel +39 (0)321 375855, Fax 39 (0)321 375621, e-mail: silvio.aprile@uniupo.it
ORCID:
Silvio Aprile:
0000-0003-4804-9543
0000-0003-1965-9748
0000-0001-6968-6176
0000-0002-2398-5944
0000-0002-1562-556X
Rosanna Matucci:
Cristina Bellucci:
Erika Del Grosso:
Giorgio Grosa:
KEYWORDS
Oxybutynin; N-oxidation; in vitro-in vivo metabolism; mass spectrometry

ABSTRACT
1.
Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an
overactive bladder and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic
metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic
effects.
2.
We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography tandem
mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring
plasma and urine samples collected after oral administration of oxybutynin in rats. This
investigation highlighted that not only N-deethylation, but also N-oxidation participates in the
clearance of oxybutynin after oral administration.
3.
A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative
metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine
function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement
of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring.
3.
demonstrating its lack of antimuscarinic activity.
4. Despite the presence of the α,β-unsaturated function, Oxy-EK does not react with
The functional activity of Oxy-EK was investigated on the muscarinic receptors (M1-3)
glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic
metabolites were formed.

INTRODUCTION
Antimuscarinics are the mainstay of pharmacological treatment of overactive bladder (OAB), a
complex disorder defined by the presence of urinary urgency, usually associated with frequency and
nocturia, with or without urinary urge incontinence. Oxybutynin hydrochloride (4-diethylamino)
but-2-ynyl(RS)-2-cyclohexyl-2-hydroxy-2-phenyl-acetate hydrochloride, Oxy) is an antimuscarinic
agent administered as a racemic mixture and is widely prescribed to OAB patients with symptoms
of urge incontinence. However, its antimuscarinic activity resides predominantly on the (R)-
enantiomer, which binds selectively to muscarinic M1 and M3 receptors in the detrusor muscle.
Today, different types of oxybutynin formulations are available, including immediate and extended
release tablets, oral solutions, syrup and a solution for intravesical administration.
Oxybutynin is absorbed from the gastrointestinal tract following oral administration, with a
systemic bioavailability of less than 10% when compared to intravenous dosing due to extensive
pre-systemic metabolism. The circulating primary metabolite, N-desethyloxybutynin (Oxy-DE), is
present in plasma at concentrations approximately 4-10 times those of the parent compound, while
having anticholinergic effect similar to oxybutynin (Douchamps,et al., 1988 and Hughes et al.,
1992). In therapeutic use, Oxy-DE appears to contribute greatly to the treatment-limiting
anticholinergic side effects associated with the oral administration of oxybutynin (Zobrist et al,
2001). Consequently, to mitigate the systemic adverse effects occurring during therapy, topical
formulations, such as an adhesive patch, were employed exploiting oxybutynin free base as the drug
substance (Shaw et al., 2007, Mizushima et al., 2007, and Staskin et al., 2010).
In the past few decades, the metabolic fate of oxybutynin and its effect on drug metabolizing
enzymes have been investigated in rats (Akimoto et al., 1986), dog, and human after oral
administration (Shinozaki et al., 1986). The authors reported the formation of a multitude of
metabolites in dog and human urine and estimated that up to ~ 30% of the administered dose was
excreted as metabolites in urine; they also observed the highest metabolic activity in rat liver. Four
metabolic pathways were identified: N-deethylation, hydrolysis of ester, hydroxylation at 3' and 4'

positions on the cyclohexane ring and the conjugation of the corresponding metabolites. In the same
year Akimoto et al., (1986) reported that, cyclohexylmandelic acid (CHMA) is the main metabolite
in rat plasma after the oral administration of oxybutynin, while the glycine conjugate is the main
metabolite in bile and urine. More recently, carboxylesterase 1 (hCE1) was identified as the major
isozyme responsible for oxybutynin hydrolysis (Sato et al., 2012). In 1998, Yaïch and co-workers
(Yaïch et al., 1998) demonstrated that the CYP3A4 subfamily is involved in the formation of the
Oxy-DE metabolite. Finally, Lindeke et al. (1981) first reported N-oxidation of the tertiary
propargylamine moiety in rat liver microsomes using deuterated substrate and GC-MS analysis.
However, they were not able to directly reveal the presence of the N-oxide metabolite (Oxy-NO),
but detected its degradation products promoted by high temperature GC-MS analysis. Indeed, they
proposed the formation of propenal and Schiff base derivatives by Oxy-NO rearrangement.
To the best of our knowledge, no further study has addressed the oxidative metabolic fate of
oxybutynin in the last thirty years. In our previous report (Canavesi et al., 2016), we elucidated the
chemical degradation pathway of oxybutynin free base in oxidative conditions; disclosing the role
of oxybutynin N-oxide in the formation of oxybutynin enaminoketone (Oxy-EK), the final
degradation product whose structure and mechanism of formation we established. In this paper, we
revisit the oxidative metabolic fate of oxybutynin in rat and human liver microsomes and in vivo in
rats, providing evidence for metabolic N-oxidation followed by rearrangement to Oxy-EK. We also
explored the possible muscarinic profile of Oxy-EK, with the hypothesis that Oxy-EK binds to
muscarinic receptor subtypes being tested in Chinese hamster ovary (CHO) cells stably expressing
M1-3 receptors.

EXPERIMENTAL
Reagents and chemicals
Acrylonitrile, atropine sulfate salt monohydrate, 1-chloroethyl chloroformate, m-chloroperbenzoic
acid (mCPBA), methanol – HPLC grade, triethylamine, and uridine 5'-diphosphoglucuronic acid
trisodium salt (UDPGA), were purchased from Sigma-Aldrich (Milano, Italy) or Alfa Aesar
(Lancashire, UK). Adenosine 3'-phosphate 5'-phosphosulfate lithium salt hydrate – 80% (PAPS)
and reduced glutathione (GSH) were purchased from Merck (Milano, Italy). [3H]N-
methylscopolamine chloride specific activity range 2,590–3,200 GBq/mmol and UniFilter GF/C
plates from Perkin-Elmer Life and Analytical Science, Monza, Milano, Italy. Water (HPLC grade)
was obtained from Milli-Q reverse osmosis system (Millipore Co., Billerica MA, USA). Column
chromatography was performed on silica gel 60 (230-400 mesh ASTM Merck). Thin layer
chromatography (TLC) was carried out on plates with a layer thickness of 0.25 mm (silica gel 60
F254; Merck); when necessary, they were visualized after spraying with ninhydrin reagent.
Oxybutynin (potency: 99.3%), was obtained as gift sample from Pharmafar S.r.l. (Torino, Italy).
Oxybutynin free base, Oxy-NO, and Oxy-EK were prepared according to the procedures previously
reported (Canavesi et al., 2016).
Instrumentation and chromatographic conditions
LC-UV analyses
A Shimadzu HPLC system (Shimadzu, Kyoto, Japan), consisting of two LC-10AD Vp module
pumps, a SLC-10A Vp system controller, a SIL-10AD Vp autosampler and a DGU-14-A on-line
degasser, was used for the analysis. All the chromatographic separations were performed on a
Kinetex C18 XB 150  4.6 mm (5 μm d.p.) column as a stationary phase protected by a C18-
Security Guard™ (Phenomenex, Torrance, CA). The SPD-M10Avp photodiode array detector was
used to detect the analytes at 210 and 311 nm. LC Solution 1.24 software was used to process the
chromatograms. Aliquots (20 μl) of the supernatants obtained from the incubations were injected

onto the HPLC system. The isocratic mobile phase (flow rate 1.4 ml/min) was composed of eluant
A ammonium acetate buffer (20 mM, pH=5.7) and eluant B methanol, with the A:B ratio being
35:65 (v/v) (total run time: 15 min). The eluants were filtered through a 0.45 μm pore size PVDF
membrane filter prior to use. All of the analyses were carried out at room temperature.
LC-MSn analyses
A Thermo Finningan LCQ Deca XP plus system equipped with a quaternary pump, a Surveyor AS
autosampler, a Surveyor photodiode array detector and a vacuum degasser was used for LC-MS
analyses (Thermo Electron Corporation, Waltham, MA). All the chromatographic separations were
performed on a Kinetex C18 50  2.10 mm (2.6 μm d.p.) column protected by a C18-Security
Guard™ (Phenomenex, Torrance, CA) kept at a 35 °C. Aliquots (5 μl) of supernatants obtained
from incubations were injected onto the system and eluted with a mobile phase (flow rate 0.2
ml/min) consisting of: A ammonium acetate buffer (10 mM, pH 5.7), and eluant B methanol. The
following gradient elution was used: 0 to 9.5 min [A=55-20%, B=45-80%], 9.5 to 10 min [A=20-
55%, B=80-45%], 10 to 15 min [A=55%, B=45%]. The eluants were filtered through a 0.45-μm
pore size polyvinylidene difluoride membrane filter before use. The eluate was injected into the
electrospray ion source (ESI) and MS spectra were acquired and processed using Xcalibur®
software. The operating conditions of the ion trap mass spectrometer were as follows: positive
mode, spray voltage, 5.00 kV; source current, 80 μA; capillary temperature, 300 °C; capillary
voltage, 22.00 V; tube lens offset, 20.00 V; multipole 1 offset, -4.25 V; multipole 2 offset, -8.00 V;
sheath gas flow (N2), 35 Auxiliary Units. Negative mode: spray voltage, 3.30 kV; source current, 80
μA; capillary temperature, 300°C; capillary voltage, -34.00 V; tube lens offset, -35.00 V; multipole
1 offset, 6.25 V; multipole 2 offset, 10.50V; sheath gas flow (N2), 50 Auxiliary Units. Data were
acquired in full-scan and product ion scan modes (MSn) using mass scan range m/z 105-700. The
collision energy was optimized at 28-30%.

Spectroscopic analyses
1H and 13C experiments were performed at 298 K on a JEOL Eclipse ECP 300 FT MHz
spectrometer (Jeol Ltd. Tokyo, Japan) operating at 7.05T. Chemical shifts are reported in part per
million (ppm).
Synthesis of the reference compounds
Benzeneacetic acid, α-cyclohexyl-α-hydroxy-4-(ethylamino)-2-butyn-1-yl ester (Oxy-DE)
Oxybutynin free base (1.32 g, 3.70 mmol) was dissolved in freshly prepared anhydrous
dichloromethane (30 ml) and 1-chloroethyl chloroformate (2.22 ml, 20.6 mmol) was added
dropwise maintaining the reaction temperature at 0°C under magnetic stirring. The reaction mixture
was further heated to reflux for 1.5 hours, and after cooling at room temperature, the solvent was
evaporated and the crude residue re-dissolved in methanol (30 ml) and heated to reflux for an
additional 1.5 hours. Evaporation of the organic solvent under reduced pressure gave a yellow oil
which was analyzed by TLC (dichloromethane/methanol 95:5, visualized with ninhydrin) obtaining
three spots: (unreacted oxybutynin: Rf =0.5 red spot, Oxy-DE: Rf =0.2 yellow spot, unknown
product: Rf =0.1 purple spot). The residue was purified by column chromatography using
dichloromethane/methanol 95:5 and then 9:1 as eluant to give Oxy-DE as pale yellow oil (295 mg,
0.90 mmol, 24% yield, HPLC purity at 210 nm 95.8%), (Supplemental material Fig. 1).
ESI-MS: calculated for C20H27NO3 mol.wt.=329.4; found m/z 330 [M+H]+.
Benzeneacetic acid, α-cyclohexyl-α-hydroxy-4-(ethylamino)-4-(hydroxy)- 2-butyn-1-yl ester (Oxy-
HA)
Oxy-DE (350 mg, 1.06 mmol) was dissolved in 5 ml of methanol in the presence of a catalytic
amount of triethylamine. A small amount of water was then added to the mixture without leading
the solution to be opalescent. Acrylonitrile (105 mg, 2.0 mmol) was added dropwise and the
mixture was stirred at room temperature for five hours. The solvent was then evaporated under

reduced pressure giving benzeneacetic acid, α-cyclohexyl-α-hydroxy-4-(ethylamino)-4-
(cyanoethyl)-2-butyn-1-yl ester, as a yellow oil (~300 mg) which was used directly without further
purification.
This crude intermediate (~300 mg, ~0.78mmol) was dissolved in few millilitres of freshly prepared
anhydrous dichloromethane and mCPBA (270 mg, 1.29 mmol, dissolved in dichloromethane), was
added dropwise maintaining the reaction at -78°C under nitrogen for 3 hours. The reaction was
further stirred at 0°C for 2 hours. After cooling at room temperature, the reaction mixture was
washed with 10% aqueous sodium carbonate solution. The organic layer was dried over anhydrous
sodium sulfate, filtered and evaporated under reduced pressure giving a crude oil which was first
purified by column chromatography (dichloromethane/methanol 98:2) and then by a second column
(petroleum ether/2-propanol 9:1) to give Oxy-HA as a pale yellow oil (85 mg, 0.25 mmol, 32 %
yield, 97.3% purity by HPLC assay), (Supplemental material Fig. 1).
ESI-MS: calculated for C20H27NO4 mol.wt.=345.4; found m/z 346 [M+H]+.
1H-NMR (300 MHz, CDCl3) δ 7.64-7.30(m, 5H-aromatic), 4.80(d, J=13.4 Hz, O-CH2-C≡), 4.75(d,
J=13.4 Hz, O-CH2-C≡), 3.59(s, ≡CH2-N), 2.75(q, J=7.14 Hz, N-CH2), 2.24-1.09 (m, CH3 and 11H-
cyclohexyl).
Rat and human liver preparations
Male Wistar Han rat liver microsomes (RLM), (protein concentration: 20 mg/ml, total P450: 650
pmol/mg protein), human liver microsomes (HLM), (pooled mixed sex, fifty individual donors,
protein concentration: 20 mg/ml, total P450: 360 pmol/mg protein), and human liver cytosol (HLC),
(pooled mixed sex, fifty individual donors, protein concentration: 20 mg/ml, sulfotransferase: 0.63
nmol/mg protein) were purchased from Corning B.V. Life Sciences (Amsterdam, The Netherlands).
All the incubations were performed using a horizontal DUBNOFF shaking thermostatic bath (Dese
Lab Research, Padova, Italy) while protecting from light.

Phase I incubations with RLM and HLM
The standard incubation mixture (0.5 ml final volume), was carried out in a 50 mM
tris[hydroxymethyl]aminomethane (TRIS)/HCl buffer (pH 7.4) containing 3.3 mM MgCl2, 1.3 mM
NADPNa2, 3.3 mM glucose 6-phosphate, 0.4 Units/ml glucose 6-phosphate dehydrogenase, 5 μl of
acetonitrile (1% of total volume), and oxybutynin (200 μM). After pre-equilibration of the mixture,
an appropriate volume of RLM or HLM suspension was added to give a final protein concentration
of 1.0 mg/ml. The mixture was shaken for 60 minutes at 37°C. Control incubations were carried out
without the presence of NADPH-regenerating system or microsomes. Each incubation was stopped
by the addition of 0.5 ml ice-cold acetonitrile, vortexed and centrifuged at 12,500 rpm for 10 min.
The supernatants were analyzed by LC-UV and LC-MS analyses. Alternatively, when the chemical
reactivity of the metabolites was studied, GSH (3mM) was added to the incubation mixture. When
Oxy-HA and Oxy-EK were studied as substrates, the incubations followed the same procedure
reported above.
Phase II incubation HLM or HLC
Glucuronide
The standard incubation mixture, constituted by 2 mM UDPGA cofactor, in a 50 mM TRIS/HCl
buffer (pH 7.4), containing 5 μl of acetonitrile (1% of total volume), oxybutynin or Oxy-HA (200
μM) was brought to a final volume of 0.5 ml. After pre equilibration of the mixture at 37 °C, an
appropriate volume of HLM suspension, previously activated by Brij 58 surfactant (0.5 mg/mg
protein) in ice for 15 min, was added to give a final protein concentration of 1.0 mg/ml. The
incubation mixture was shaken for 60 minutes at 37°C. Control incubations were carried out in the
absence of HLM or UDPGA cofactor. The incubations were stopped by addition of 0.5 ml ice-cold
acetonitrile, vortexed and centrifuged at 12,500 rpm for 10 min prior to LC-MS analyses of the
supernatants.

Sulfate
The standard incubation mixture, constituted by 0.1 mM PAPS cofactor, in a 50 mM TRIS/HCl
buffer (pH 7.4), containing 5 μl of acetonitrile (1% of total volume), and compound Oxy-HA,
brought to a final volume of 0.5 ml. After pre-equilibration of the mixture at 37 °C, an appropriate
volume of HLC suspension was added to give a final protein concentration of 1.5 mg/ml. The
incubation mixture was shaken for 60 minutes at 37°C. Control incubation was carried out in the
absence of PAPS. Each incubation was stopped by addition of 0.5 ml ice-cold acetonitrile, vortexed
and centrifuged at 12,500 rpm for 5 min. The supernatants were analyzed by LC-MS.
In vivo rat metabolism
Animals
Male Sprague Dawley (8-9 weeks, 250-300 g) rats were purchased from Envigo S.r.l. (Bresso,
Italy). Animal studies were performed in accordance with EU and institutional guidelines approved
by the
Bioethics Committee for Animal Experimentation of the University of Eastern Piedmont, Italy
(Prot. DB064.11 - 14th September 2016). The animals were housed with free access to food and
water and fasted for 16 h before administration. All the rats were maintained under a 12 h cycle of
light/dark artificial lighting at 21-25 °C.
Oxybutynin administration and sample collection
Oxybutynin hydrochloride (75 mg/Kg) was administered to rats (n=8) by oral gavage (1.5 ml),
dissolving the dose in saline. Blood samples were collected via the tail vein in heparinized tubes 1
and 2 h following the administration anesthetizing the rats with isoflurane. Urine samples were
collected over the periods 0-6, 6-24 h and kept cool by placing the containers in ice. Blood samples
were immediately centrifuged to obtain the plasma samples. All samples were stored at -80 °C until
the analysis.

Determination of oxybutynin metabolites in rat plasma and urine
Preparation of calibration standards
Stock solutions of oxybutynin, Oxy-DE, Oxy-EK, and Oxy-HA were prepared in methanol at 1
mg/ml concentration. Standard working solutions were freshly obtained by serial dilution of the
stock solutions with methanol. The calibration standards were prepared by spiking appropriate
aliquots of working solutions (5µl) into blank plasma or urine (45 µl) and diluting with 100 or 50 µl
of ice-cold acetonitrile respectively. Calibrators were homogenized and centrifuged at 12,500 rpm
for 10 min. Supernatants (10 µl) were injected into the LC-MS system. The calibration curves (y =
ax + b), were constructed from the peak areas versus analyte concentration (over a range of 5-1000
µg/l) using the weighted (1/x) linear least-squares regression method. Precision and accuracy were
determined by repeated analyses (n = 3) of the five calibrators used to construct the calibration
curve for each analyte. Precision was expressed as percent coefficient of variation (%CV), whereas
the accuracy was determined as the percent relative error (%RE) of the mean back calculated
concentrations from the theoretical concentrations. The mean precision (%CV) for all the analytes
was ≤ 20%, whereas the mean accuracy (%RE) were 11.1%, 18.1%, 4.1%, and 17.8% for
Oxybutynin, Oxy-DE, Oxy-EK and Oxy-HA respectively.
Plasma and urine samples preparation
Aliquots of plasma and urine samples (50 µl) were diluted by adding respectively 100 or 50 µl of
ice-cold acetonitrile. Samples were homogenized and centrifuged at 12,500 rpm for 10 min.
Supernatants (10 µl) were injected into the LC-MS system.
In vitro pharmacological characterization of Oxy-EK in human muscarinic receptor subtypes:
displacement of [3H]-N-methylscopolamine
3H-NMS competition binding assays were carried out in polypropylene 96-well plates, using
membranes from (CHO) cells stably expressing recombinant human M1-M3 muscarinic

acetylcholine receptors (mACh) receptors as previously described (Matucci et al., 2016). In general,
membrane homogenates (30-70 μg/ml, final concentration) were incubated in 0.25 ml total volume
of assay buffer (25 mM sodium phosphate containing 5 mM MgCl2 at pH 7.4) containing [3H]-
NMS (0.2 nM) and a range of concentrations of oxybutynin and Oxy-EK at room temperature for
120 min; nonspecific binding was determined in the presence of 1 μM atropine. Receptor bound
radioactivity was separated by filtration using UniFilter GF/C plates and was measured by
scintillation counting. Results are expressed as means ± SEM of the percentage of inhibition (I%) of
specific binding at 10 µM concentrations.
RESULTS
In vitro metabolism study
Phase I incubations with RLM and HLM
The in vitro metabolic fate of oxybutynin was assessed through its incubation in rat and human liver
subcellular preparations in the presence or absence of a NADPH-regenerating system. The
incubation samples were analyzed by both LC-UV and LC-MS to obtain data on the metabolic
stability and the structures of the metabolites respectively. When the microsomal mixed-function
oxidase system was activated by the presence of NADPH, oxybutynin underwent a relevant
oxidative transformation with the residual substrate ranging from 10% (RLM) to 56% (HLM)
(Supplemental material Fig. 2, 3). Similar values were obtained in incubations supplied with 3mM
GSH with the residual substrate ranging from 15% (RLM) to 62% (HLM).
Structural characterization of oxybutynin metabolites was achieved by LC-MS tandem mass
spectrometry analysis of the incubations carried out with HLM. All the metabolites were
characterized based on their predicted mass shift from the oxybutynin protonated molecule at m/z
358, and the interpretation of their MSn spectra. Oxybutynin incubation samples were analyzed in
both positive full scan and product ion scan modes, revealing the phase I metabolites pattern
reported in the chromatograms in Fig.1. As a matter of fact, the presence of eleven metabolite-

related ions was observed, whose MS2 spectra and proposed fragmentation pathways are depicted in
Fig. 2 and 3. Monitoring the ion at m/z 374, LC-MS2 chromatogram revealed the presence of six
peaks corresponding to the following metabolites: M1-M3 eluting at 3.37, 3.84, and 5.10 min
respectively, Oxy-NO (7.15 min), Oxy-EK (9.52 min), and a minor metabolite M7 eluting at 4.60
min (Fig. 1).
Metabolites M1, M2, and M3 showed identical product ion spectra; among them, the MS2 spectrum
of M1 is reported in Fig. 3. For these metabolites, the protonated molecule at m/z 374 gave rise to a
fragmentation pathway similar to that observed for oxybutynin. Indeed, the fragment ions at m/z
124, 142, and 258 correspond to those observed in the oxybutynin spectrum, whereas the fragments
at m/z 187, 283 and 356 presented a shift of 16 Da with respect to the same ions found in the
oxybutynin spectrum. Overall, this set of data suggested that the M1, M2, and M3 metabolites arose
from enzymatic oxidation on the cyclohexane ring.
Oxy-NO product ion spectrum (m/z 374) gave two diagnostic fragment ions at m/z 189 and 171,
corresponding to the same ions observed in the oxybutynin MS2 spectrum (Fig. 3). Moreover, the
presence of the fragment ions at m/z 356 (loss of water), and 158 (ester cleavage), both shifted by
16 Da with respect to the same ions in the oxybutynin spectrum clearly demonstrated that the
enzymatic oxidation occurred on the propargylamine moiety, leading to the formation of N-oxide
metabolite.
The structure of Oxy-NO was unequivocally confirmed by comparing its mass spectrum with that
of synthetic Oxy-NO provided from the oxidation of oxybutynin free base with hydrogen peroxide
(Canavesi et al., 2016).
Oxy-EK protonated molecules (m/z 374) generated a two-fragment MS2 spectrum (Fig. 2). The
most abundant ion (m/z 356) was due to the neutral loss of water whereas the ion at m/z 158
(generated from the ester cleavage) corresponded to the oxybutynin fragment ion at m/z 142 shifted
by 16 Da. Considered together, these data indicated that the structure of Oxy-EK is identical to that
of the degradation product previously reported in the chemical stability study of oxybutynin

(Canavesi et al.,2016). Even if the structure of Oxy-EK presents a potentially electrophilic α,β-
unsaturated ketone function, its formation was not affected by the addition of GSH to the
incubation, indicating the stability of the enaminoketone moiety.
The product ion spectrum of the minor metabolite M7 (m/z 374) gave similar fragmentation
pathways to that of Oxy-NO (Supplemental material Fig. 4). The presence of the ions at m/z 158
(shifted of 16 Da respect to the same ion at m/z 142 in oxybutynin spectrum), and m/z 189 clearly
indicated that the addition of an oxygen atom occurred on the N,N-diethyl propargylamino moiety.
However, these data are insufficient to propose a structure for this metabolite.
The relevant literature currently states that Oxy-DE is the circulating primary metabolite after
oxybutynin oral administration. It is characterized by a protonated molecule at m/z 330 whose
fragmentation generated the ions at m/z 312 (loss of water) and m/z 230 (100 Da neutral fragment
loss) which corresponded to the ion at m/z 258 observed in the oxybutynin spectrum. In liver
microsomes, Oxy-DE underwent further oxidative N-deethylation generating the primary amine
metabolite M6. The structure assignment was based on the presence of the protonated molecule at
m/z 302, whose fragmentation generated the ions at m/z 284 (loss of water) and at m/z 202 (100 Da
neutral fragment) which corresponded to the ion at m/z 230 observed in the Oxy-DE spectrum.
Moreover, Oxy-DE underwent hydroxylation at the cyclohexane ring (metabolites M4 and M5) and
N-oxidation generating the corresponding hydroxylamine Oxy-HA. In particular, metabolites M4
and M5 gave identical MS2 spectra, as featured by the presence of the diagnostic ions at m/z 187,
215, and 283 (the same ions were found in the M1 spectrum), and the ion at m/z 230, resulting in
the neutral loss of a 116 Da fragment. This fragmentation pattern indicated that the metabolites M4
and M5 arose from aliphatic hydroxylation on the cyclohexane ring. The secondary metabolite Oxy-
HA gave a protonated molecule at m/z 346; the presence of fragment ions at m/z 171, 229 and 246
(loss of a 100 Da neutral fragment) indicated that oxidation on the propargylamine moiety had
occurred. The structure of Oxy-HA was unequivocally confirmed by comparison of its retention
time and fragmentation pathways with those of a synthetic standard. The formation of Oxy-HA was

observed when synthetic Oxy-DE was incubated in the same conditions as oxybutynin, confirming
that it arises from the oxidation of secondary amino group. Finally, according to the results of Sato
et al. (2012), the formation of the CHMA metabolite arising from the hydrolysis of ester function
was observed in this study, confirming the presence of the ion at m/z 233 [M-H]- (data not shown).
Phase II incubations.
Glucuronidation and sulfation.
In order to assess the formation of glucuronide metabolites, HLM incubations of oxybutynin and
Oxy-HA were carried out in the presence of UDPGA cofactor. The formation of sulfates was
evaluated by incubating the same substrates in the presence of HLC fractions and the PAPS
cofactor. All of the incubations were analyzed by LC-MS in negative ionization mode.
The formation of a metabolite eluting at 4.04 min was observed in the Oxy-HA incubation in the
presence of UDPGA, while the same peak is absent in control incubations. The metabolite
generated a deprotonated molecule at m/z 520, corresponding to the addition of 175 Da with respect
to the parent compound, suggesting the formation of an Oxy-HA glucuronide (Fig.4). This
attribution was further supported by the presence of the diagnostic fragment ion at 344 m/z (neutral
loss of glucuronic acid) as well as the presence of additional fragments at m/z 502, 304 and 233 m/z.
However, the formation of Oxy-HA glucuronide was not observed in oxybutynin incubation.
Similarly, the formation of oxybutynin glucuronide was not observed due to the lack of the
corresponding ion at m/z 532 (data not shown). Finally, in the incubation of Oxy-HA performed in
HLC supplied by PAPS, the absence of peaks in the chromatogram monitoring the negative ion at
m/z 424 indicated that sulfate metabolites were not formed (data not shown).
In vivo rat metabolism.
To assess the in vivo formation of the new metabolites Oxy-EK and Oxy-HA, a study was
undertaken in rats. The analysis of plasma samples, collected 1 and 2 h following oxybutynin

administration, revealed the presence of the parent drug and its metabolites Oxy-DE, Oxy-EK, but
the absence of Oxy-HA and its glucuronide (Supplemental Fig. 5-7). The same metabolites were
observed in urine samples collected over the periods 0-6 h and 6-24 h. after the treatment
(Supplemental Fig. 6-7). The availability of the synthetic reference compounds for Oxy-DE and
Oxy-EK (Supplemental Fig. 8) allowed their quantification. In plasma samples, oxybutynin was
detected at concentrations ranging from 34 to 431 µg/l, while both Oxy-DE and Oxy-EK were
present in comparable amounts at a concentration lower than 10 µg/l. In the urine samples, the
mean concentration of oxybutynin was lower with respect to plasma concentrations, while both
Oxy-DE and Oxy-EK were present at concentrations up to 100 µg/l (Fig. 5).
Due to the intrinsic instability of Oxy-NO synthetic standard (Canavesi et al., 2016), only the
qualitative evaluation of Oxy-NO metabolite was provided. As a result, a prominent peak related to
the presence of Oxy-NO was revealed in both plasma and urine samples at each time point
(Supplemental Fig. 9). The stability of Oxy-NO and its rearrangement to enaminoketone were also
investigated in rat plasma and urine samples spiked with a freshly prepared solution of Oxy-NO
(Supplemental Fig. 10A). Overall, the obtained stability profiles indicated the progressive
conversion over time of Oxy-NO to Oxy-EK: the residual Oxy-NO ranges from 86%, when
incubated in plasma for 2 h at 37°C, to 13%, when incubated in urine for 24 h at 37°C
(Supplemental Fig. 10B). Interestingly, Oxy-NO resulted stabilized in spiked plasma and urine
samples compared to the control incubations carried out in methanol. Finally, the relative amount of
Oxy-NO and Oxy-EK resulted unchanged in the spiked samples stored at-80°C for 24h
demonstrating the role of temperature in the Oxy-NO interconversion to Oxy-EK.

In vitro pharmacological characterization of Oxy-EK in human muscarinic receptor subtypes.
3H-NMS competition binding assays, using membranes from CHO cells stably expressing the
recombinant human M1-M3 mACh, revealed that Oxy-EK was devoid of significant affinity toward
the receptors (Tab. 1).
DISCUSSION
Depending on their structural features, the metabolic stability of drugs could be strictly related to
their chemical stability; hence, the integration of both aspects could be conveniently performed
defining a unique chemical and metabolic stability space. Using this viewpoint, we re-investigated
the metabolic fate of oxybutynin by exploiting the recent findings in its chemical stability, both in
vitro (rat and human liver subcellular preparations) and in vivo in rats, focusing our attention on the
oxidative transformation of the tertiary propargylamine amine moiety.
The results allowed us to delineate a new metabolic scheme for oxybutynin (Fig. 6), observing a
certain regio-selectivity, with only the tertiary amine and the cyclohexyl moieties being oxidized.
Three distinct oxidative metabolic pathways were observed, namely: N-deethylation (Oxy-DE and
M6), N-oxidation (Oxy-NO and Oxy-HA), and the aliphatic hydroxylation on cyclohexane ring for
both oxybutynin (M1-M3) and Oxy-DE (M4-M5). The N-oxidation of oxybutynin was previously
proposed by Lindeke et al. (1981); however, due to the thermal instability of N-oxide, it could not
be directly detected using high-temperature GC-MS analysis. In our study, we detected Oxy-NO by
means of LC-MSn analysis in rat and human liver microsomal incubations unequivocally assigning
its structure by comparison of its spectral data with those of the authentic N-oxide generated by
oxidation of oxybutynin with hydrogen peroxide. It is worth mentioning that Oxy-NO was found in
a significant amount in urine, indicating that the rearrangement could not be so relevant in the
context of the metabolic fate of oxybutynin in physiological conditions.

The presence of the unprecedented metabolite Oxy-EK was also observed, while its formation
through the previously described intramolecular prototropic rearrangement of oxybutynin N-oxide,
(Szabó and Hermecz, 2001, Canavesi et al., 2016), was proposed (Supplemental Fig. 11).
Another noteworthy aspect is the chemical and metabolic stability of Oxy-EK; no metabolite was
generated from it when incubated as a substrate in rat and human liver microsomes. Despite the
presence of an α,β-unsaturated keto-function, no chemical and enzyme-mediated reactivity (i.e.
gluthatione S-transferases) with GSH was observed. These results corroborate with our previous
data, where the presence of the α,β-unsaturated function on Oxy-EK does not give rise to genotoxic
potential (Canavesi et al, 2016). These results are in agreement with our previous data dealing with
the lack of genotoxicity of Oxy-EK as observed in Ames assay. These features could be explained
by the lack of basicity of nitrogen in the enaminoketone moiety, and by the push-pull character of
the central double bond that would impose a significant presence of a mesomeric and dipolar form.
This would mitigate the Michael acceptor property of the α,β-unsaturated function.
Since Oxy-DE represents a primary metabolite found in significant amounts both in vitro and in
vivo experiments, the N-oxidation of the secondary amine function to Oxy-HA and the N-
deethylation to the primary amino metabolite M6, could be expected. Accordingly, the presence of
the new metabolite Oxy-HA was observed in liver microsomal incubations and confirmed by means
of the synthetic reference compound. Moreover, the formation of the corresponding glucuronide
was also established after the incubation of Oxy-HA. Conversely, analysis of the plasma and urine
samples excluded the presence of significant amounts of Oxy-HA and its glucuronide. This might
not be surprising, considering that Oxy-HA was solely a minor in vitro metabolite of oxybutynin
and the related glucuronide was only detected after Oxy-HA incubation with liver microsomes.
The literature shows that oxybutynin is well absorbed from the gastrointestinal tract following oral
administration, but that its systemic bioavailability is less than 10% when compared to intravenous
dosing due to extensive pre-systemic metabolism. The circulating primary metabolite, Oxy-DE, was
found in plasma at concentrations approximately 4–10 times than those of the parent compound and

has similar anticholinergic effects to oxybutynin (Hughes et al., 1992, Zobrist et al., 2001). Based
on our in vivo investigation, we demonstrated the presence of the parent drug and its metabolites
Oxy-DE, Oxy-EK, and Oxy-NO in rat plasma and urine, following oral administration of Oxy.
Taken together, both the in vitro and in vivo data demonstrated that Oxy-DE, Oxy-NO and Oxy-EK
all participate in the clearance of oxybutynin after oral administration.
To assess the pharmacodynamics of the new metabolites, the activity of the chemically stable
metabolite, Oxy-EK, on the three main muscarinic receptor subtypes (hM1-3, widely expressed in
peripheral systems), was investigated. The enaminoketone function of Oxy-EK could not be
protonated at physiological pH (which differs from the propargylamine moiety of Oxy), mitigating
its affinity with muscarinic cholinergic receptor, and accordingly, the experimental data showed that
Oxy-EK is devoid of antimuscarinic activity.
In conclusion, taking together the in vitro and in vivo data, we highlighted that both the N-
deethylation and N-oxidation metabolic pathways participate in the clearance of oxybutynin after
oral administration. Moreover, we demonstrated the lack of antimuscarinic activity of the newly
reported metabolite Oxy-EK, and the presence of the α,β-unsaturated function did not give rise to a
genotoxic potential indicating that the clearance of oxybutynin does not produce an active or
putatively toxic metabolite.
ACKNOWLEDGEMENTS
Financial support from the University of Eastern Piedmont, Novara, Italy, (MedChem-2015), is
gratefully acknowledged.
DECLARATION OF INTEREST
The authors report no declarations of interest. The authors alone are responsible for the content and
writing of this article.

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FIGURE CAPTIONS
Figure 1. Positive LC-MS2 chromatograms of oxybutynin incubation in HLM.
Figure 2. LC-MS2 spectra and fragmentation pathways of oxybutynin and Oxy-DE, Oxy-HA, and
Oxy-EK metabolites.
Figure 3. LC-MS2 spectra and fragmentation pathways of M1, M4, M6, and Oxy-NO metabolites.
Figure 4. LC-MS2 chromatogram and fragmentation pathways of Oxy-HA glucuronide metabolite.
Figure 5. Mean plasma and urine concentrations of oxybutynin, Oxy-DE, and Oxy-EK. Oral
administration of oxybutynin hydrochloride in male rats at a dose of 75 mg/Kg (mean ± S.E.M.,
n=8).
Figure 6. Proposed in vitro, in vivo metabolic pathways of oxybutynin.

TABLES
Table 1. Experimental values of binding data for oxybutynin and Oxy-EK with different human
mACh receptors subtypes.
I%
compound
hM1
hM2
hM3
Oxy
99.19 ± 0.53
99.55 ± 0.45
99.90 ± 0.10
Oxy-EK
10.27 ± 0.66
6.15 ± 1.25
28.15 ± 4.35
Percentage of inhibition (I%) of specific binding at 10 μM concentration.Values are means ±
S.E.M. from three separate determinations in duplicate.