aldehyde oxidase activity in fresh human...

DMD Manuscript #60368

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Aldehyde oxidase activity in fresh human skin

Nenad Manevski, Kamal Kumar Balavenkatraman, Barbara Bertschi, Piet Swart, Markus Walles,

Gian Camenisch, Hilmar Schiller, Olivier Kretz, Barbara Ling, Reto Wettstein, Dirk J. Schaefer,

Francois Pognan, Armin Wolf, and Karine Litherland

Drug Metabolism and Pharmacokinetics (NM, PS, MW, GC, HS, OK, and KL), Pre-clinical

Safety (KKB, BB, FP, and AW), Novartis Institutes for BioMedical Research, Novartis Pharma,

Basel, Switzerland. Department of Plastic, Reconstructive, Aesthetic and Hand Surgery,

University Hospital Basel, Switzerland (BL, RW, DJS)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on September 23, 2014 as DOI: 10.1124/dmd.114.060368

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Running title: Aldehyde oxidase activity in human skin explants

Corresponding author:

Karine Litherland, PhD Novartis Institute for Biomedical Research Translational Sciences/DMPK/IDD (Integrated Drug Disposition) Fabrikstrasse 14-1.02.7 Novartis Pharma AG CH-4002 Basel, Switzerland Phone: +41797226137 E-mail: [email protected]

Numbers:

Number of text pages: 20 Number of tables: 3 Number of figures: 6 Number of references: 54 Number of words in Abstract: 250 Number of words in Introduction: 560 Number of words in Discussion: 1491

List of nonstandard abbreviations:

AO, aldehyde oxidase; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; NATs, N-

acetyltransferases; SULTs, sulfotransferases; UGTs, UDP-glucuronosyltransferases.


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Abstract

Human aldehyde oxidase (AO) is a molybdo-flavoenzyme that commonly oxidizes

azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in

several drug failures, existing in vitro–in vivo correlations are often poor and extrahepatic role of

AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin,

the largest organ of the body frequently exposed to therapeutic drugs and xenobiotics. Fresh full-

thickness human skin was obtained from 13 individual donors and assayed with two specific AO

substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized

carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of

carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol.mg skin–1.h–1, resulting in

13% and 2% of substrate turnover after 24 h of incubation with 10 μM of substrate, respectively.

Hydroxylation activities for the two substrates were significantly correlated (r2 = 0.769), with

interindividual variability ranging from 3 (zoniporide) to 6-fold (carbazeran). Inclusion of

hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of

hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 μM of

the inhibitor. Reaction rates were linear up to 4 h and well described by Michaelis-Menten

enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan

glucuronidation and sulfation, and p-toluidine N-acetylation, showed that cutaneous AO activity

is comparable to tested phase II metabolic reactions, indicating a significant role of AO in

cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic

activity in human skin.


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Introduction

Aldehyde oxidase (AO) is a molybdo-flavoenzyme that oxidizes electrophilic carbons of

azaheterocycles, such as pyridine, pyrimidine, and pyridazine, scaffolds often included in

therapeutic drugs to increase solubility, lower logP, and avoid cytochrome P450 (CYP)-mediated

metabolism (Garattini and Terao, 2013; Hutzler et al., 2013). In addition, as the enzyme name

suggests, AO oxidizes aldehydes to carboxylic acids, a role probably related to conversion of

endogenous retinaldehyde (retinal) into retinoic acid (Terao et al., 2009, Graessler and Fischer,

2007). Although AO drug metabolism is relevant for pharmacotherapy, many researchers failed

to predict high in vivo AO metabolic clearance based on in vitro results, leading to notable drug

failures such as carbazeran (Kaye et al., 1984), BIBX1382 (Dittrich et al., 2002), zoniporide

(Dalvie et al., 2010), RO1 (Zhang et al., 2011), SGX523 (Diamond et al., 2010), and FK3453

(Akabane et al., 2011). Difficulties in predicting AO clearance probably arise from several

confounding factors: (i) cytosolic enzyme localization (Kaye et al., 1985), (ii) apparent enzyme

instability in the in vitro assay systems (Duley et al., 1985, Al-Salmy, 2001, Hutzler et al., 2014),

(iii) large interindividual (Hutzler et al., 2014) and interspecies differences (Dalvie et al., 2013),

and (iv) potential contribution of extrahepatic tissues, most notably kidneys (Nishimura and

Naito, 2006) and respiratory tissues (Moriwaki et al., 2001). Taken together, further research is

needed to better understand and characterize AO drug metabolism, especially its tissue

localization, suitable in vitro experimental systems, and methods that would aid in vitro–in vivo

extrapolation.

Human skin, the largest organ of the body, is frequently exposed to therapeutic drugs, either after

topical and transdermal administration, or following drug distribution from the systemic

circulation. Besides pharmacotherapy, daily life also exposes skin to numerous cosmetic

ingredients and environmental xenobiotics, many of which share structural features with drugs


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and may cause drug interactions. Since biotransformation in human skin can lead to metabolic

elimination of drugs, as well as to their activation or toxification (Sharma et al., 2013),

understanding cutaneous drug metabolism is becoming increasingly important for drug discovery,

safety, and development (Gundert-Remy et al., 2014, Gotz et al., 2012b, Gotz et al., 2012a, Jackh

et al., 2011). Although AO was semi-quantitatively detected in the human skin at the level of

mRNA (Hu et al., 2010) and protein (van Eijl et al., 2012), cutaneous enzyme activity of AO

remains unknown. This knowledge gap may negatively impact drug development projects,

especially if skin is the administration site or target tissue for therapy. In addition to drug

development, general lack of knowledge about the cutaneous AO metabolism could obstruct

cosmetics development and, as was recently exemplified by the case of AO-mediated SGX523

toxification (Diamond et al., 2010), impede our general understanding of adverse drug reactions

in the skin.

To address the open questions of AO biotransformation in human skin, this study investigated

AO enzymatic activity in the fresh full-thickness human skin explants, an experimental model

that contains all relevant cell types and intact skin morphology (Lebonvallet et al., 2010).

Enzyme activities of two specific AO substrates, carbazeran (Kaye et al., 1984, Kaye et al., 1985)

and zoniporide (Dalvie et al., 2010), were tested in healthy skin from 13 donors, also shedding

light on interindividual variability. Results reveal, for the first time, that human skin possesses

significant AO activity, with reaction rates comparable to other more established cutaneous

eliminations routes, for example glucuronidation, sulfation, and N-acetylation.


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Materials and Methods

Reagents and chemicals

Belzer UW cold storage solution (organ preservation medium) was obtained from Bridge to Life

(Columbia, SC). Cytotoxicity detection kit plus, a lactate dehydrogenase release assay, was

acquired from Roche (Basel, Switzerland). Penicillin-streptomycin-glutamine (10,000 U·mL–1

penicillin, 10,000 μg·mL–1 streptomycin, 29.2 mg·mL–1 glutamine; used as 100-fold dilution) was

purchased from Life Technologies (Carlsbad, CA). Williams E medium (1.8 mM Ca2+, no

glutamine), dimethyl sulfoxide (DMSO, ≤99.7%), insulin from bovine pancreas (≥27 USP

units·mg–1), hydrocortisone (suitable for cell culture), formic acid (≥98%), p-toluidine (99.7%),

4’-methylacetanilide (99%), carbazeran (≥96%), zoniporide hydrochloride hydrate (≥98%),

17β-estradiol-3-β-D-glucuronide sodium salt (≥98%), hydralazine hydrochloride (99%), and in

vitro toxicology assay kit (MTT based) were purchased from Sigma-Aldrich (Buchs,

Switzerland). The 4-hydroxycarbazeran, 2-oxo-zoniporide hydrochloride, triclosan O-sulfate

sodium salt, and triclosan O-β-D-glucuronide sodium salt were acquired from Toronto Research

Chemicals (Toronto, Canada). Organic solvents of LC-MS or higher purity grade were used in

this study. Stock solutions of carbazeran, zoniporide, 4’-methylacetanilide (internal standard for

LC-MS analysis), triclosan, and p-toluidine were prepared in DMSO (10–30 mM) and stored at –

20 °C until use. Stock solution of hydralazine hydrochloride, an irreversible inhibitor of AO, was

prepared in methanol : water (50:50, v/v; 25 mM) and also stored at –20°C until use.

Materials for tissue explant culture

Stericup® filter units (0.22 μm) and receiver flasks were purchased from Millipore (Billerica,

MA). Sterile biopsy punch tools, 4 mm in diameter, were ordered from Stiefel (GlaxoSmithKline

company, Research Triangle Park, NC). Falcon® 24-well plates were purchased from Corning

(Tewksbury, MA).


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Skin explant culture

The collection of fresh healthy human skin surgical waste samples was performed at the Institute

for Plastic, Reconstructive, Aesthetic and Hand Surgery, Basel University Hospital, Switzerland,

in accordance with the Declaration of Helsinki (1964 and subsequent revisions). Local ethics

committee of Basel, Switzerland, approved the study protocol. Skin donors gave written informed

consent before entering the study (13 adult subjects, 41–72 years old. See Table 1 for further

demographics information). Human skin tissue excised during the surgery was collected and

placed in bottles filled with sterile Belzer UW organ preservation solution and supplemented with

penicillin (100 Units·mL–1) and streptomycin (100 μg·mL–1). Human skin was transported to our

laboratories at 4°C, usually within 30–90 min from the time of surgical excision.

Once in our laboratory, skin tissue was handled under aseptic condition and processed with sterile

dissection tools. After removing adipose tissue and hypodermis with surgical scissors, cylindrical

skin explants, 4 mm in diameter, were prepared using a sterile skin biopsy tools. Skin thickness

varied based on anatomical region and individual characteristics of the donor (generally 1–2 mm

for breast, inguinal, and axillary skin; 2–5 mm for abdomen and thigh skin). If the skin was

thicker than 3 mm, dermis was trimmed with curved surgical scissors to achieve maximal overall

skin thickness of approximately 3 mm. Prepared skin explants were placed in pre-warmed

Williams E medium supplemented with insulin (10 μg·mL–1), hydrocortisone (10 ng·mL–1), and

penicillin-streptomycin-glutamine (100 Units·mL–1 of penicillin; 100 μg·mL–1 of streptomycin; 2

mM L-glutamine) (Lu et al., 2007). Culturing temperature was 37°C, incubator humidity was

90%, and CO2 content was 5%. During method development, skin explant viability after 24 h of

culture was confirmed by histology (hematoxylin and eosin staining), lactate dehydrogenase

release, and MTT viability assays.


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Drug metabolism assays

Human skin explants were incubated in 500 μL of Williams E medium spiked with test

compounds. Skin incubations were performed in duplicate. Concentrations of carbazeran and

zoniporide were 10 μM, except in enzyme kinetics assays (0.5–30 μM). Incubation time for

screening with multiple skin donors and inhibition assays was 15–24 h. For time-course assays,

formation of metabolites was monitored from 1 to 24 h. Enzyme kinetics assays were performed

at incubation time of both 1 and 4 h. Final DMSO concentration was 0.1%. If hydralazine was

included in the assays, the medium also contained 0.05% of methanol. These concentrations of

organic solvents had minimal impact on AO activity measured in human liver cytosol (Behera et

al., 2014). To maximally expose skin tissue to probe substrates and inhibitors, skin explants were

freely floating in the medium with the epidermis facing the air-liquid interface. After incubation

time, both skin explants and corresponding incubation media were placed in centrifuge tubes and

snap-frozen in liquid nitrogen. Samples were stored at –80°C until metabolite extraction and

analysis. All incubations were performed in duplicate. Negative control samples were prepared in

parallel: (i) probe substrates in Williams E medium without skin explants and (ii) skin explants in

Williams E medium without probe substrate.

Analytics and data analysis

To extract metabolites from the skin explants, skin tissue was first crushed with a cryoPREP™

Impactor (Covaris, Woburn, MA). Skin explants were placed in TT05XT tissue tubes (Covaris,

Woburn, MA), cooled in liquid nitrogen, and then hammered once with cryoPREP™ Impactor

(impact strength 2). Crushed skin was transferred to centrifuge tubes filled with lysing matrix D

(MP Biomedicals, Santa Ana, CA), 1 mL of 70% acetonitrile was added (v/v), and samples were

additionally homogenized using a FastPrep instrument (MP Biomedicals, Santa Ana, CA;

agitation speed was 4.0 m·s–1; three cycles of 20 seconds each). The combination and cryo-


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hammer and ceramic beads impact resulted in complete homogenization of the skin tissue. Skin

incubation medium was directly mixed with acetonitrile (30:70 v/v) and vortexed for 1 min. All

samples were kept overnight at –20°C, centrifuged at 30,000g for 30 min, and aliquots of the

supernatants were spiked with 500 nM of suitable internal standard (Table 2). Samples were

evaporated to dryness in vacuum and then reconstituted with mobile phase. Skin extraction

procedure was previously developed and optimized with Novartis compounds in development

(data not shown).

After extraction from skin explants and corresponding incubation medium, drug metabolites were

detected and quantified using Quattro Ultima triple-quadruple mass spectrometer with electro-

spray source (Waters, Milford, MA), coupled with an Agilent 1100 capillary HPLC pump

(Agilent, Santa Clara, CA), and CTC Pal autosampler (CTC Analytics, Zwingen, Switzerland).

Metabolite quantification was performed relative to internal standards in multiple-reaction

monitoring mode and resulting chromatograms were analyzed with MassLynx 4.1 software

(Waters, Milford, MA). For all methods, eluents A and B were 0.1% formic acid in water and

0.1% formic acid in acetonitrile, respectively. Analytes were separated on the following HPLC

columns: (A) Agilent StableBond® C18 (50 × 1.0 mm, 3.5 μm, Agilent, Santa Clara, CA), (B)

Agilent StableBond® C18 (150 × 1.0 mm, 3.5 μm), and (C) Phenomenex Luna®

pentafluorophenyl (150 × 1.0 mm, 3 μm, Phenomenex, Torrance, CA). Eluent flow rate was 0.1

mL/min, column temperature was 40 °C, and injection volume was 10 μL. Lower limits of

detection and quantification were estimated based on signal-to-noise ratios of 3 and 10,

respectively. To prepare standard curves, mobile phase was spiked with authentic metabolite

standards in the concentration range of 1–1000 nM and suitable internal standards (Table 2).

Metabolite extraction procedure and chromatographic methods were optimized to minimize the

matrix effect in reconstructed skin extracts (as judged by signal intensity of the internal standards,


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matrix effects were relatively minor, up to 20% of signal intensity decrease compared to samples

prepared in mobile phase). Further details of the analytical methods are presented in Table 2.

Amounts of metabolites quantified were normalized to average skin punch weight (for the

individual donor) and total media volume (500 μL). To calculate the total amount of metabolite

formed in the incubation, the amounts quantified in skin punch extract and corresponding

medium extract were summed up. Enzyme kinetic parameters were obtained by fitting the

Michaelis-Menten model (� ��

��

��

) to the experimental data, using GraphPad Prism version

6.02 for Windows (GraphPad Software Inc., San Diego, CA, USA). The v is a reaction velocity,

Vmax is the limiting reaction velocity, Km is the Michaelis-Menten constant, and [S] is the total

concentration of substrate.


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Results

Collection and culturing of human skin samples

Fresh healthy full-thickness human skin samples were acquired from 13 individual donors

undergoing cosmetic or reconstructive surgery. Although donors were predominantly females

(85%) of the Caucasian origin (77%), study also included two males and one female of black

origin (Table 1). Skin samples were obtained from different anatomical regions, namely from

abdomen (6 donors), breast (3 donors), inguinal area (3 donors), and axilla (1 donor). Median

donor age was 55 years, ranging from 41 to 72 years. To preserve viability of the tissue, excised

human skin was transported in the Belzer UW solution, a specialized medium particularly

developed for solid organ transplantation (Sollinger et al., 1989). Skin culturing conditions were

optimized prior to drug metabolism assays, namely the preparation of skin explants, volume and

composition of the incubation medium, calcium concentration, and the use of antibiotics.

Histological analysis of formalin-fixed paraffin embedded skin tissue, stained with hematoxylin

and eosin, demonstrated that skin explants kept normal macroscopic morphology and viability at

least for the first 24 h. After optimization of skin culturing conditions, drug metabolism

incubations were routinely initiated within 30–90 min from the time of surgical excision.

Activity of aldehyde oxidase in the human skin explants

To investigate the activity of AO in human skin, the hydroxylation of two selective AO substrates,

carbazeran and zoniporide, was measured in full-thickness skin explants. Because stratum

corneum may limit the penetration of compounds into the tissue, test substrates were directly

added to the incubation medium. Initial assays were performed at 10 μM concentration of

substrate and incubation times ranging from 15 to 24 h. To better understand interindividual

variability in AO activities, skin incubations were performed with skin explants from 13


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individual donors in total, 12 with carbazeran and 10 with zoniporide (skin from one donor was

used only for enzyme kinetics assays at 4 h incubation time).

Results showed that all tested skin samples hydroxylated carbazeran to 4-hydroxycarbazeran and

zoniporide to 2-oxo-zoniporide (Fig. 2, Table 1). Metabolites were absent in the negative controls:

(i) Williams E medium spiked with substrate but without skin explants and (ii) skin explants

incubated in Williams E medium without any added substrate. Average measured activity rates

for carbazeran 4-hydroxylation, expressed as pmol of formed product per mg of skin tissue and

incubation time (pmol·mg skin–1·h–1), were approximately 10-fold higher than corresponding

activity rates of zoniporide 2-hydroxylation (Fig. 2A, see Table 1 for activities of individual

donors and corresponding summary statistics). Interindividual variability of carbazeran

hydroxylation (6-fold) was also higher than the corresponding variability of zoniporide

hydroxylation (3-fold). Activities of carbazeran and zoniporide hydroxylation were not

significantly correlated with donors’ demographic data, namely gender, age, race, anatomical

region, smoking, or alcohol intake (data now shown). If substrate turnover is calculated based on

metabolite formation, assuming that AO-metabolism is the only metabolic pathway, average

substrate turnover for carbazeran and zoniporide hydroxylation was 13 and 2%, respectively (Fig.

2B). As shown by the Pearson’s correlation coefficient (r = 0.877, r2 = 0.769, p = 0.0009), the

hydroxylation activities of the two substrates were significantly positively correlated (Fig. 2C).

Correlation test was based on the assumption that both carbazeran and zoniporide hydroxylation

activities were sampled from a population following the Gaussian distribution.

Inhibition of carbazeran and zoniporide hydroxylation

To confirm the major role of AO in the cutaneous metabolism of carbazeran and zoniporide,

inhibition assays with hydralazine, an irreversible inhibitor of AO, were performed. Initial assays

were performed at 10 μM of substrate and 25 μM of hydralazine, without any pre-incubation


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steps (Figs. 3A and 3B). Although statistically significant inhibition levels were achieved, 25 μM

of hydralazine failed to completely abolish the carbazeran and zoniporide hydroxylation activities.

Subsequent assays were performed at the higher concentrations of the hydralazine, 50 and 100

μM, also including the 1 h pre-incubation with hydralazine in order to prevent substrate

protection from enzyme inactivation (Fig. 3C). Results demonstrate that 100 μM of hydralazine

diminished activities of carbazeran 4-hydroxylation by more than 90%, thus supporting the major

role of AO.

Time-course of carbazeran and zoniporide hydroxylation in human skin explants

To estimate the linearity of metabolite formation over incubation time, carbazeran and zoniporide

hydroxylations were tested during the initial 24 h of the skin explant culture (Fig. 4). Although

the hydroxylation of both substrates was linear up to approximately 4 h of incubation, reaction

rates progressively decelerated afterwards. These results indicate that reaction rates for 24 h

incubations, calculated as pmol·mg skin–1·h–1, underestimated initial AO enzyme activity. Even if

incubations times for several days are unsuitable for detailed mechanistic assays, 24 h

incubations enabled accurate quantification of a small amount of newly formed metabolites, a

benefit especially relevant for assays with zoniporide.

During the time-course experiment, the distribution of newly formed metabolites

(4-hydroxycarbazeran and 2-oxo-zoniporide) differed between skin explants and corresponding

medium (Fig. 4). After 1 h of incubation, majority of metabolites were extracted from the skin

explant. However, at later time points, metabolites were predominantly found in the incubation

medium.

Enzyme kinetics of carbazeran and zoniporide hydroxylations in human skin explants

To determine the enzyme kinetic parameters of carbazeran and zoniporide hydroxylations in the

human skin explants, incubations were performed at eight different substrate concentrations,


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ranging from 0.5 to 30 μM (Fig. 5, Table 3). Higher concentrations were avoided because of

possible impact on skin explant viability and necessity for higher percentage of organic solvent.

In light of the results obtained from the time-course assays (Fig. 4), enzyme kinetics was

performed at both 4 and 24 h of incubation time (Fig. 5). Since extracellular and intracellular

concentrations of substrates may significantly differ in the skin explant model, derived enzyme

kinetic parameters were designated as apparent values (Km,app and Vmax,app). Hydroxylation of both

substrates was well described by the hyperbolic Michaelis-Menten model, with coefficients of

determination (r2) ranging from 0.95 to 0.97 (Table 3). Although Km,app values were unaffected

by the incubation time, limiting reaction velocities Vmax,app were considerably higher after 4 h

incubation. This finding reflects deviation from the reaction rate linearity observed during the

time-course assays (Fig. 4). The Km,app value of carbazeran hydroxylation (approx. 3.5 μM) was

significantly lower than the corresponding apparent Km,app value of zoniporide hydroxylation

(approx. 21 μM).

Comparison of carbazeran and zoniporide hydroxylation with cutaneous glucuronidation and sulfation of triclosan, and N-acetylation of p-toluidine

Compared to phase I metabolic enzymes, existing literature suggested higher expression levels of

phase II metabolic enzymes in human skin (Hu et al., 2010, van Eijl et al., 2012, Luu-The et al.,

2009), especially UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), and N-

acetyltransferases (NATs). To compare the cutaneous activities of AO with corresponding

reaction rates of UGTs, SULTs, and NATs, carbazeran and zoniporide hydroxylations were

assayed in parallel with triclosan glucuronidation and sulfation, and p-toluidine N-acetylation

(Fig. 6). Triclosan and p-toluidine were selected as high activity substrates for the corresponding

phase II reactions, based on preliminary assays that included several substrates for


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glucuronidation, sulfation, and N-acetylation (data not shown, separate manuscript in preparation).

All incubations were performed for 24 h at 10 μM of substrate.

Results in skin explants from donor 10 showed that activity rate of carbazeran 4-hydroxylation is

comparable to rates of triclosan glucuronidation and sulfation, but approximately 3-fold lower

than rates of p-toluidine N-acetylation (Fig. 6). On the other hand, rates of zoniporide 2-

hydroxylation were considerably lower than all other tested reactions (Fig. 6). This comparison

demonstrated that cutaneous AO activities can be comparable to activities of cutaneous phase II

metabolic reactions, especially if the chemical structure of the AO substrate is favorable, such as

in the case of carbazeran.


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Discussion

Although AO has been known to oxidize nitrogen-containing heterocycles for a long time (Knox,

1946, Stanulovic and Chaykin, 1971, Stubley et al., 1979), its importance to drug metabolism

first emerged in the case of carbazeran, a phosphodiesterase-2 inhibitor discontinued due to low

oral bioavailability and short half-life in humans (Kaye et al., 1984, Kaye et al., 1985). Based on

analysis of chemical structure, a recent review suggested that a large number of drugs on the

market (approx. 13%) or candidate drugs (almost 45% of drugs in development) could be AO

substrates (Pryde et al., 2010). With the increasing role of AO in drug metabolism, ongoing

research activities focused exclusively on the human liver (Hutzler et al., 2014, Hutzler et al.,

2012, Obach et al., 2004), even if gene expression (Nishimura and Naito, 2006) and

immunohistochemistry (Moriwaki et al., 2001) studies suggested a considerable extrahepatic

presence, especially in kidneys and lungs. Moreover, efforts to predict AO clearance in vivo

based on in vitro assays in hepatic experimental models generally resulted in underestimation

(Hutzler et al., 2012, Zientek et al., 2010). The mRNA and protein expressions of AO were

recently also detected in human skin (Hu et al., 2010, van Eijl et al., 2012), but its activity to date

remains unknown. Since human skin is frequently exposed to therapeutic drugs, environmental

xenobiotics, and cosmetic ingredients, it is important to understand its potential for

biotransformation of drugs and drug-like compounds.

To investigate the activity of AO in the human skin, two reactions selectively catalyzed by AO

were studied: carbazeran 4-hydroxylation (Kaye et al., 1984, Kaye et al., 1985) and zoniporide 2-

hydroxylation (Dalvie et al., 2010, Dalvie et al., 2013, Dalvie et al., 2012) (Fig. 1). All assays

were performed in fresh full-thickness human skin explants, an experimental model that not only

contains all relevant skin cell types and unaltered gene expression, but also avoids risks of

enzyme inactivation during the freeze-thaw cycles and homogenization of fibrous skin tissue


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(Lebonvallet et al., 2010). Since aldehyde oxidase is known for high interindividual variability

(Hutzler et al., 2014), carbazeran and zoniporide hydroxylations were tested in skin of 12 and 10

individual donors, respectively (see Table 1 for information about donor demographics).

Combined with sensitive LC-MS analytics (Table 2), this proof-of-concept study offered a unique

opportunity to quantify AO activity in the human skin.

The results revealed that all tested human skin samples hydroxylated both carbazeran and

zoniporide (Fig. 2, Table 1). Carbazeran 4-hydroxylation activities were approximately 10-fold

higher relative to zoniporide, with apparent substrate turnover reaching almost 20% for some

donors (Fig. 2B, 10 μM incubations for 24 h). Higher cutaneous activity of carbazeran

hydroxylation is consistent with higher scaled intrinsic clearance measured previously in human

liver cytosol [carbazeran 323 mL·min–1·kg–1, zoniporide 37 mL·min–1·kg–1 (Zientek et al., 2010)].

Activities of carbazeran and zoniporide hydroxylation also showed a significant positive

correlation for 10 donors tested together (Fig. 2C), a result expected for two substrates that are

metabolized by the single human aldehyde oxidase enzyme (Garattini and Terao, 2013, Hutzler et

al., 2013). To our best knowledge, this is the first report of AO enzyme activity in the human

skin. Terao et al. (2009) recently reported activity of AO homolog 2 in mice skin, whereas Ueda

et al. (2005) investigated AO catalyzed reduction of nitro polycyclic aromatic hydrocarbons in

skin samples from hamster, rabbit, guinea pig, mouse, and rat. Although the expression of AO

was also reported in human adipose tissue (Weigert et al., 2008), the underlying fat was carefully

removed from tested human skin samples, thus eliminating the possibility for a misinterpretation

of results.

Interindividual variability between the slowest and the fastest metabolizer was 6- and 3-fold for

carbazeran and zoniporide hydroxylations, respectively (Table 1). This variability was similar to


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4.25-fold difference reported in a set of cryopreserved hepatocytes from 5 donors (Sahi et al.,

2008), but considerably lower than 17-fold variation recently observed for O6-benzylguanine

hydroxylation in cryopreserved hepatocytes of 75 donors (Hutzler et al., 2014) or 18-fold

variation in N-[(2-dimethylamino)ethyl]acridine-4-carboxamide clearance by liver cytosol of 13

donors (Al-Salmy, 2001). Observed interindividual variability is likely caused by the differences

in the expression levels of human AO or by a number of single nucleotide polymorphisms

recently observed in human AOX1 gene (Hartmann et al., 2012). The relatively robust activity of

AO in the human skin, despite the differences in donor demographics and skin’s anatomical

region, may be related to physiological role of AO in the metabolism of endogenous retinoids

(Terao et al., 2009, Graessler and Fischer, 2007, Tomita et al., 1993). A recent study in AO

homolog 2–/– knockout mice observed thickening of the epidermis in basal conditions and after

UV light exposure, probably triggered by local deficiency of all-trans retinoic acid (Terao et al.,

2009).

Hydralazine is an irreversible inhibitor of AO (Johnson et al., 1985, Strelevitz et al., 2012) that

has minor inhibitory effects on activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4/5 at

concentrations up to 50 μM (Strelevitz et al., 2012). Even if raloxifene was reported as the most

potent inhibitor of AO (Ki = 2.9 nM) (Obach et al., 2004), it is also known to inactivate CYP3A4

(Chen et al., 2002), thus lacking the preferred inhibition selectivity. In addition, high

concentrations of hydralazine have only modest effects on cellular growth (Evenson and

Fasbender, 1988) with estimated concentration to cause 50% cytotoxicity in rat hepatocytes at 8

mM (Tafazoli and O'Brien, 2008), suggesting that incubations with skin explants are unlikely to

cause significant reduction of cell viability. Inclusion of hydralazine in carbazeran and zoniporide

skin incubations reduced the hydroxylation activities in a concentration-dependent manner,

exceeding 90% inhibition of carbazeran 4-hydroxylation with 100 μM of inhibitor (Fig. 3).


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Together with reaction specificities of carbazeran 4-hydroxylation (Kaye et al., 1985) and

zoniporide 2-hydroxylation (Dalvie et al., 2013), potent inhibition by hydralazine offers an

additional evidence for cutaneous activity of aldehyde oxidase.

Reaction rates of carbazeran and zoniporide hydroxylation in skin explants decelerated after 4 h,

suggesting that activities for 24 h incubations, expressed as pmol·mg skin–1·h–1, are

underestimated (Fig. 4). Although skin histology showed normal microscopic morphology after

24 h, deviation from linearity is expected due to substrate consumption, metabolite accumulation,

or both. For example, Kaye et al. (1985) found an inhibitory effects of 4-hydroxycarbazeran on

carbazeran hydroxylation in liver cytosol. Interestingly, the predominant extracellular localization

of metabolites after 24 h indicated that 4-hydroxycarbazeran and 2-oxo-zoniporide are excreted

from the skin cells, either by passive diffusion or active transport. Osman-Ponchet et al. (2014)

recently reported expression and activity of a number of efflux transporters in the human skin,

most notably multidrug resistance-associated protein 1 (MRP1/ABCC2).

Rates of cutaneous carbazeran and zoniporide hydroxylation increased hyperbolically with

increasing substrate concentrations (Fig. 5). Apparent substrate affinities (Km,app), as derived from

the Michaelis-Menten model, were independent of the incubation time, but reaction limiting

velocities (Vmax,app) were significantly higher for the 4 h incubations (Table 3), also reflecting the

results of the time-course assay (Fig. 4). Considering that in assays with tissue explants

extracellular concentrations of substrates may significantly differ from the corresponding

intracellular concentrations, for example due to limited skin penetration and nonspecific tissue

binding, derived enzyme kinetic parameters should be considered as apparent values obtained in

the specific experimental model. As a result of this model limitation, measured Km,app and Vmax,app

values cannot be directly compared with literature values obtained with subcellular fractions or


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purified enzyme, at least before additional studies on substrate distribution and nonspecific

binding are performed. The Km of carbazeran 4-hydroxylation with partially purified human AO

was 40 μM (Beedham et al., 1987), whereas Dalvie et al. (2010) reported Km of 3.4 μM for

zoniporide 2-hydroxylation in pooled human liver cytosol.

Existing literature data suggests that cutaneous phase II metabolic enzymes, for example UGTs,

SULTs, and NATs (Gotz et al., 2012a, Jackh et al., 2011, Hu et al., 2010, van Eijl et al., 2012,

Luu-The et al., 2009, Kushida et al., 2011, Bonifas et al., 2010b, Bonifas et al., 2010a), have

higher expression and activity levels compared to corresponding cutaneous phase I metabolic

enzymes (Gotz et al., 2012b, Jackh et al., 2011). To compare the cutaneous activity of AO with

the known cutaneous phase II reactions, this study tested the glucuronidation and sulfation of

triclosan (Moss et al., 2000), and N-acetylation of p-toluidine (Gotz et al., 2012a), together with

hydroxylations of carbazeran and zoniporide. As shown in Fig. 6, activity of carbazeran

hydroxylation was comparable to triclosan glucuronidation and sulfation and approximately

3-fold lower than p-toluidine N-acetylation, thus indicating a potential significant contribution of

AO to cutaneous biotransformation, especially for high-affinity substrates.

In conclusion, to our best knowledge, this study is the first report of AO drug metabolism in the

human skin. Relatively high carbazeran activities with substrate turnover up to 20%, robust

activity with low interindividual variation, and reaction rates comparable to phase II reactions all

indicate that AO may have an important role in cutaneous drug metabolism and homeostasis.

This finding has direct implications not only for topically and transdermally administrated drugs,

but also for therapeutics that may distribute into the skin from the systemic circulation. Further

research efforts will be needed to identify specific skin cells that have the highest AO activities,


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as well as to estimate the contribution of cutaneous AO activity to total metabolic clearance of

drugs predominantly eliminated by AO.


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Acknowledgements

Authors gratefully acknowledge Marie-Catherine Stutz, Karine Bigot, Arno Doelemeyer, and

Armelle Grevot for their help with skin histology. Authors also express gratitude to Bertrand-Luc

Birlinger, Judith Streckfuss, and Maxime Garnier for providing reagents and technical assistance.


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Authorship Contributions

Participated in research design: Nenad Manevski, Karine Litherland, Hilmar Schiller, Kamal

Kumar Balavenkatraman

Conducted experiments: Nenad Manevski, Barbara Bertschi, Barbara Ling

Contributed new reagents or analytical tools: Nenad Manevski, Barbara Bertschi

Performed data analysis: Nenad Manevski

Wrote or contributed to the writing of the manuscript: Nenad Manevski, Karine Litherland,

Kamal Kumar Balavenkatraman, Barbara Ling, Olivier Kretz, Francois Pognan, Hilmar Schiller,

Gian Camenisch, Markus Walles, Piet Swart, Reto Wettstein, Dirk J. Schaefer, Armin Wolf


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Legends for Figures

Figure 1. Aldehyde oxidase catalyzes hydroxylation of carbazeran to 4-hydroxycarbazeran (A)

and zoniporide to 2-oxo-zoniporide (B).

Figure 2. (A) Rates of aldehyde oxidase activity in the human skin explants. Incubation time

was 15–24 h. Results are also presented in Table 1. (B) Apparent substrate turnover after 15–24 h

of incubation. Values are calculated based on metabolite formation, assuming that AO-catalyzed

hydroxylations are the only metabolic pathway. (C) Correlation between hydroxylations of

carbazeran and zoniporide in the human skin explants. Pearson correlation coefficient r = 0.877,

coefficient of determination r2 = 0.769, p = 0.0009 (two-tailed).

Figure 3. Inhibition of carbazeran 4-hydroxylation (A) and zoniporide 2-hydroxylation (B) by 25

μM of hydralazine (irreversible AO inhibitor). (C) Inhibition of carbazeran 4-hydroxylation by 50

and 100 μM of hydralazine (assay included 1 h pre-incubation with inhibitor). Incubation time

was 24 h. Results are presented as the mean value and S.D. for each donor. Statistical

significance was calculated by the unpaired Student’s t-test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p

≤0.001; n.s., not significant).

Figure 4. Time-course of carbazeran 4-hydroxylation (A) and zoniporide 2-hydroxylation (B) in

the full-thickness human skin explants. Assays are performed with human skin from donor 7.

Results are presented as the mean value and S.D. In addition to total metabolite formed (circles),

each panel also presents metabolite quantified in either skin punches (squares) or corresponding

incubation medium (triangles).

Figure 5. Enzyme kinetics of carbazeran 4-hydroxylation (panels A1 and A2) and zoniporide

2-hydroxylation (panels B1 and B2) in the full-thickness human skin explants. The 24 h

incubations (A1 and B1) are performed with skin from donor 8, whereas 4 h incubations are


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performed with skin from donor 11. Activities are presented as the mean value and S.D. Activity

data were fitted to Michaelis-Menten model. Calculated enzyme kinetics parameters are

presented in Table 3.

Figure 6. Comparison of activity rates for carbazeran 4-hydroxylation, zoniporide

2-hydroxylation, triclosan glucuronidation, triclosan sulfation, and p-toluidine N-acetylation in

the full-thickness human skin explants from donor 10. Measured activities are presented as the

mean value and S.D.


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Tables

Table 1. Demographics of skin donors participating in the study and activities of carbazeran 4-hydroxylation and zoniporide 2-hydroxylation measured in the corresponding human skin explants. Metabolites formed in incubations, 4-hydroxycarbazeran and 2-oxo-zoniporide, were detected and quantified in both skin punch and corresponding medium.

Skin donor

Gender Race Age Skin

anatomical region

Smoking Alcohol

Carbazeran 4-hydroxylation Zoniporide 2-hydroxylation

Mean SD Mean SD

pmol·mg–1·h–1 pmol·mg–1·h–1

1 Female Caucasian 72 Abdomen Never Occasionally 0.337 0.040 0.082 0.004

2 Female Not specified 57 Breast Never Never 1.985 0.043 0.213 0.005

3 Male Caucasian 45 Abdomen Daily Occasionally 1.429 0.031 0.165 0.004

4 Female Caucasian 46 Abdomen Never Never 1.058 0.018 0.125 0.010

5 Female Caucasian 63 Inguinal Never Occasionally 1.364 0.299 0.152 0.006

6 Female Not specified 58 Abdomen Never Never 1.481 0.020 0.157 0.029

7 Female Caucasian 47 Breast Never Occasionally 1.430 0.023 0.176 0.016

8 Female Caucasian 72 Breast Never Occasionally 1.244 0.064 0.153 0.010

9 Female Caucasian 41 Abdomen Daily Occasionally 1.952 0.181 0.217 0.009

10 Female Black 45 Axillary Occasionally Occasionally 1.248 0.144 0.210 0.016

11 Female Caucasian 52 Inguinal Daily Occasionally 1.256 0.023 — —

12 Male Caucasian 55 Abdomen Never Occasionally 0.789 0.069 — —

13 Female Caucasian 55 Inguinal Former 1–2 Days/week * * * *

Summary statistics:

Number of donors tested: 12 10

Average activity rates (± S.D.): 1.301 ± 0.449 0.164 ± 0.043

Coefficient of variation: 34% 26%

Activity range (min–max): 0.337–1.985 0.082–0.217 —, not tested *, enzyme kinetics assays only

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on September 23, 2014 as D

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d.114.060368 at ASPET Journals on May 7, 2018 dmd.aspetjournals.org Downloaded from



Table 2. Analytical methods used for separation, detection, and quantification of metabolites formed in the human skin explants. Abbreviations: LOD, lower limit of detection; LOQ, lower limit of quantification.

Analyte Column Gradient Internal standard Ionization Transitions Capillary Voltage/

Cone/Collision

LOD/ LOQ

m/z kV/V/V nM

4-Hydroxycarbazeran A

0–5 min, 5→95% B; 5–10 min, 95% B; 10–10.1

min, 95→5% B; and 10.1–17 min, 5% B.

4’-Methylacetanilide ESI+ 377>234 377>288

2.5/30/18 <1

2-Oxo-zoniporide C

0–2 min, 5→95% B; 2–12 min, 95% B; 12–12.1 min, 95→5% B; 12.1–20

min, 5% B

4’-Methylacetanilide ESI+ 337>236 337>250 337>278

2.5/50/16 <1 / 2.1

4’-Methylacetanilide B

0–5 min, 5→95% B; 5–14 min, 95% B; 14–14.1 min, 95→5% B; 14.1–22

min, 5% B

4-Hydroxycarbazeran ESI+ 150>93 150>108

2.5/30/20 <1

Triclosan O-sulfate

A

0–1 min, 10% B; 1–5 min, 10→95% B; 5–15 min, 95% B; 15–15.1

min, 95→10% B; 15.1–22 min, 10% B

17β-Estradiol-3-β-D-glucuronide

ESI–

367>287 369>289

2.5/20/13 <1

Triclosan O-β-D-glucuronide

463>287 465>289

2.5/20/13 <1


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Table 3. Apparent enzyme kinetic parameters of carbazeran 4-hydroxylation and zoniporide 2-hydroxylation in the full-thickness human skin explants. Assays were performed for both 4 and 24 h. Activity data were fitted to Michaelis-Menten model. Results are presented as the best fit value ± standard error of the mean.

Carbazeran 4-hydroxylation Zoniporide 2-hydroxylation Km,app Vmax,app r2 Donor Km,app Vmax,app r2 Donor

Incubation time (h): μM pmol·mg–1·h–1 μM pmol·mg–1·h–1 24 3.57 ± 0.48 1.62 ± 0.06 0.97 8 20.71 ± 4.67 0.37 ± 0.04 0.96 8 4 3.48 ± 0.56 3.46 ± 0.16 0.95 13 21.37 ± 5.75 2.22 ± 0.33 0.95 13


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