1521-009X/42/12/2049–2057$25.00 DRUG METABOLISM AND DISPOSITION Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/dmd.114.060368 Drug Metab Dispos 42:2049–2057, December 2014

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 (N.M., P.S., M.W., G.C., H.S., O.K., K.L.) and Pre-clinical Safety (K.K.B., B.B., F.P., A.W.), Novartis Institutes for BioMedical Research, Novartis Pharma, Basel, Switzerland; and Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital Basel, Basel, Switzerland (B.L., R.W., D.J.S.) Received July 30, 2014; accepted September 22, 2014

ABSTRACT activities for the two substrates were significantly correlated (r2 = 0.769), with interindividual variability ranging from 3-fold (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 mM inhibitor. Reaction rates were linear up to 4 hours and well described by MichaelisMenten 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.

Introduction

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, after either 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 and may cause drug interactions. Because 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 (Jäckh et al., 2011; Götz et al., 2012a,b; Gundert-Remy et al., 2014). Although AO was semiquantitatively 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 affect 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 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 fresh, full-thickness

Aldehyde oxidase (AO) is a molybdoflavoenzyme that oxidizes electrophilic carbons of azaheterocycles, such as pyridine, pyrimidine, and pyridazine, scaffolds often included in therapeutic drugs to increase solubility, lower log P, and avoid cytochrome P450–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 (Graessler and Fischer, 2007; Terao et al., 2009). 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: 1) cytosolic enzyme localization (Kaye et al., 1985); 2) apparent enzyme instability in the in vitro assay systems (Duley et al., 1985; Al-Salmy, 2001; Hutzler et al., 2014); 3) large interindividual (Hutzler et al., 2014) and interspecies differences (Dalvie et al., 2013); and 4) potential contribution of extrahepatic tissues, most notably kidneys (Nishimura and Naito, 2006) and respiratory tissues (Moriwaki et al., 2001). Thus, further research is needed to better understand and characterize AO drug metabolism, especially its tissue dx.doi.org/10.1124/dmd.114.060368.

ABBREVIATIONS: AO, aldehyde oxidase; DMSO, dimethylsulfoxide; LC-MS, liquid chromatography–mass spectrometry; NAT, N-acetyltransferase; SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase. 2049

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Human aldehyde oxidase (AO) is a molybdoflavoenzyme 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 the 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% substrate turnover, respectively, after 24 hours of incubation with 10 mM substrate. Hydroxylation

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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 (Fig. 1), carbazeran (Kaye et al., 1984, 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 those of other more established cutaneous elimination routes, for example, glucuronidation, sulfation, and N-acetylation. Materials and Methods

v¼

Vmax ½S Km þ ½S

to the experimental data, using GraphPad Prism version 6.02 for Windows (GraphPad Software, Inc., La Jolla, CA). The v is a reaction velocity, Vmax is

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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 units×ml–1 penicillin, 10,000 mg×ml–1 streptomycin, and 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), dimethylsulfoxide (DMSO; #99.7%), insulin from bovine pancreas ($27 USP units×mg–1), hydrocortisone (suitable for cell culture), formic acid ($98%), p-toluidine (99.7%), 49-methylacetanilide (99%), carbazeran ($96%), zoniporide hydrochloride hydrate ($98%), 17b-estradiol-3-b-D-glucuronide sodium salt ($98%), hydralazine hydrochloride (99%), and in vitro toxicology assay kit [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide based] were purchased from Sigma-Aldrich (Buchs, Switzerland). The 4-hydroxycarbazeran, 2-oxo-zoniporide hydrochloride, triclosan O-sulfate sodium salt, and triclosan O-b-D-glucuronide sodium salt were acquired from Toronto Research Chemicals (Toronto, ON, Canada). Organic solvents of liquid chromatography–mass spectrometry (LC-MS) or higher purity grade were used in this study. Stock solutions of carbazeran, zoniporide, 49-methylacetanilide (internal standard for LC-MS analysis), triclosan, and p-toluidine were prepared in DMSO (10–30 mM) and stored at –20C 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 –20C until use. Materials for Tissue Explant Culture. Stericup filter units (0.22 mm) and receiver flasks were purchased from Millipore (Billerica, MA). Sterile biopsy punch tools, 4 mm in diameter, were ordered from Stiefel (a GlaxoSmithKline company, Research Triangle Park, NC). Falcon 24-well plates were purchased from Corning (Tewksbury, MA). 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 (Basel, Switzerland), in accordance with the Declaration of Helsinki (1964 and subsequent revisions). The local ethics committee of Basel 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 mg×ml–1). Human skin was transported to our laboratories at 4C, usually within 30–90 minutes of the time of surgical excision. Once in our laboratory, skin tissue was handled under aseptic conditions 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 sterile skin biopsy tools. Skin thickness varied based on anatomic region and individual characteristics of the donor (generally 1–2 mm for breast, inguinal, and axillary skin and 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 ;3 mm. Prepared skin explants were placed in prewarmed Williams E medium supplemented with insulin (10 mg×ml–1), hydrocortisone (10 ng×ml–1), and penicillin-streptomycin-glutamine (100 units×ml–1 of penicillin, 100 mg×ml–1 of streptomycin, and 2 mM L-glutamine) (Lu et al., 2007). Culturing temperature was 37C, incubator humidity was 90%, and CO2 content was 5%. During method development, skin explant viability after 24 hours of culture was confirmed by histology (H&E), lactate dehydrogenase release, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assays. Drug Metabolism Assays. Human skin explants were incubated in 500 ml of Williams E medium spiked with test compounds. Skin incubations were

performed in duplicate. Concentrations of carbazeran and zoniporide were 10 mM, except in enzyme kinetics assays (0.5–30 mM). Incubation time for screening with multiple skin donors and inhibition assays was 15–24 hours. For time-course assays, formation of metabolites was monitored from 1–24 hours. Enzyme kinetics assays were performed at incubation times of both 1 and 4 hours. 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 –80C until metabolite extraction and analysis. All incubations were performed in duplicate. Negative control samples were prepared in parallel: probe substrates in Williams E medium without skin explants and 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), 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 (v/v) was added, and samples were additionally homogenized using a FastPrep instrument (MP Biomedicals) at an agitation speed of 4.0 m·s–1 for three cycles of 20 seconds each. The combination of cryo-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 minute. All samples were kept overnight at –20C and centrifuged at 30,000g for 30 minutes, and aliquots of the supernatants were spiked with 500 nM suitable internal standard (Table 2). Samples were evaporated to dryness in vacuum and then reconstituted with mobile phase. The 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 triplequadrupole mass spectrometer with electrospray source (Waters, Milford, MA), coupled with an Agilent 1100 capillary high-performance liquid chromatography 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). 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 high-performance liquid chromatography columns: (A) Agilent StableBond C18 (50
1.0 mm, 3.5 mm), (B) Agilent StableBond C18 (150
1.0 mm, 3.5 mm), and (C) Phenomenex Luna pentafluorophenyl (150
1.0 mm, 3 mm) (Phenomenex, Torrance, CA). Eluent flow rate was 0.1 ml/min, column temperature was 40C, and injection volume was 10 ml. 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). The 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, matrix effects were relatively minor, up to 20% of signal intensity decrease compared with 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 ml). 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

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Aldehyde Oxidase Activity in Human Skin Explants 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. Carbazeran 4-Hydroxylation Skin Donor

Gender

Race

Age

Skin Anatomic Region

Smoking

Mean

S.D.

Caucasian Not specified Caucasian Caucasian Caucasian Not specified Caucasian Caucasian Caucasian Black Caucasian Caucasian Caucasian

72 57 45 46 63 58 47 72 41 45 52 55 55

S.D.

–1

pmol×mg skin ×h

Abdomen Breast Abdomen Abdomen Inguinal Abdomen Breast Breast Abdomen Axillary Inguinal Abdomen Inguinal

Never Never Daily Never Never Never Never Never Daily Occasionally Daily Never Former

Occasionally Never Occasionally Never Occasionally Never Occasionally Occasionally Occasionally Occasionally Occasionally Occasionally 1–2 days/wk

No. of donors tested Average (6 S.D.) activity rate (pmol×mg skin–1×h–1) Coefficient of variation Activity range (min–max) (pmol×mg skin–1×h–1)

0.337 1.985 1.429 1.058 1.364 1.481 1.430 1.244 1.952 1.248 1.256 0.789 –a

0.040 0.082 0.043 0.213 0.031 0.165 0.018 0.125 0.299 0.152 0.020 0.157 0.023 0.176 0.064 0.153 0.181 0.217 0.144 0.210 0.023 – 0.069 – a – –a Summary Statistics

0.004 0.005 0.004 0.010 0.006 0.029 0.016 0.010 0.009 0.016 – – –a

12 1.301 6 0.449

10 0.164 6 0.043

34% 0.337–1.985

26% 0.082–0.217

–, not tested. a Enzyme kinetics assays only.

the limiting reaction velocity, Km is the Michaelis-Menten constant, and [S] is the total concentration of substrate.

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 Caucasian origin (77%), the study also included two males and one female of black origin (Table 1). Skin samples were obtained from different anatomic 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 41–72 years. To preserve viability of the tissue, excised human skin was transported in 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. Histologic analysis of formalin-fixed, paraffin-embedded skin tissue, stained with H&E, demonstrated that skin explants kept normal macroscopic morphology and viability at least for the first 24 hours. After optimization of skin culturing conditions, drug metabolism incubations were routinely initiated within 30–90 minutes of the time of surgical excision. Activity of AO 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 (Fig. 1). Because the stratum corneum may limit the penetration of compounds into the tissue, test substrates were added directly to the incubation medium. Initial assays were performed at 10 mM concentration of substrate and incubation times ranging from

TABLE 2 Analytical methods used for separation, detection, and quantification of metabolites formed in the human skin explants

Analyte

Column

4-Hydroxycarbazeran

A

2-Oxo-zoniporide

C

49-Methylacetanilide

B

Triclosan O-sulfate

A

Gradient

Internal Standard

Ionization

49-Methylacetanilide

ESI+

49-Methylacetanilide

ESI+

4-Hydroxycarbazeran

ESI+

17b-Estradiol3-b-D-glucuronide

ESI–

Transitions m/z

Triclosan O-b-D-glucuronide

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

ESI, electrospray ionization; LOD, lower limit of detection; LOQ, lower limit of quantification.

377 377 337 337 337 150 150 367 369 463 465

→ → → → → → → → → → →

234 288 236 250 278 93 108 287 289 287 289

Capillary Voltage/ Cone/Collision

LOD/LOQ

kV/V/V

nM

2.5/30/18

,1

2.5/50/16

,1/2.1

2.5/30/20

,1

2.5/20/13

,1

2.5/20/13

,1

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Female Female Male Female Female Female Female Female Female Female Female Male Female

Mean –1

yr

1 2 3 4 5 6 7 8 9 10 11 12 13

Zoniporide 2-Hydroxylation

Alcohol Use

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Fig. 1. AO catalyzes hydroxylation of carbazeran to 4-hydroxycarbazeran (A) and zoniporide to 2-oxo-zoniporide (B).

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15–24 hours. To better understand interindividual variability in AO activities, skin incubations were performed with skin explants from 13 individual donors in total, 12 with carbazeran and 10 with zoniporide (skin from 1 donor was used only for enzyme kinetics assays at 4 hours’ 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: 1) Williams E medium spiked with substrate but without skin explants and 2) skin explants incubated in Williams E medium without any added substrate. Average measured activity rates for carbazeran 4-hydroxylation, expressed as picomoles of formed product per milligram of skin tissue and incubation time (pmol×mg skin–1×h–1), were ;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, anatomic region, smoking, or alcohol intake (data not 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

Fig. 2. (A) Rates of AO activity in the human skin explants. Incubation time was 15–24 hours. Results are also presented in Table 1. (B) Apparent substrate turnover after 15–24 hours 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).

Aldehyde Oxidase Activity in Human Skin Explants

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10 mM substrate and 25 mM hydralazine, without any preincubation steps (Fig. 3, A and B). Although statistically significant inhibition levels were achieved, 25 mM 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 mM, also including the 1-hour preincubation with hydralazine to prevent substrate protection from enzyme inactivation (Fig. 3C). The results demonstrate that 100 mM hydralazine diminished activities of carbazeran 4-hydroxylation by .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 hours of the skin explant culture (Fig. 4). Although the hydroxylation of both substrates was linear up to ;4 hours of incubation, reaction rates progressively decelerated afterward. These results indicate that reaction rates for 24-hour incubations, calculated as picomoles of formed product per milligram of skin tissue per hour of incubation time, underestimated initial AO enzyme activity. Even if incubation times of several days are unsuitable for detailed mechanistic assays, 24-hour 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 hour of incubation, the 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, ranging from 0.5–30 mM (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 assays were performed at both 4 and 24 hours of incubation time (Fig. 5). Because 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 0.95–0.97 (Table 3). Although Km,app values were unaffected by the incubation time, limiting reaction velocities Vmax,app were considerably higher after 4 hours of 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 (;3.5 mM) was significantly lower than the corresponding apparent Km,app value of zoniporide hydroxylation (;21 mM). Comparison of Carbazeran and Zoniporide Hydroxylation with Cutaneous Glucuronidation and Sulfation of Triclosan and N-Acetylation of p-Toluidine. Compared with phase I metabolic enzymes, existing literature suggested higher expression levels of phase II metabolic enzymes in human skin (Luu-The et al., 2009; Hu et al., 2010; van Eijl et al., 2012), 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

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Fig. 3. Inhibition of carbazeran 4-hydroxylation (A) and zoniporide 2-hydroxylation (B) by 25 mM hydralazine (irreversible AO inhibitor). (C) Inhibition of carbazeran 4-hydroxylation by 50 and 100 mM hydralazine (assay included 1-hour preincubation with inhibitor). Incubation time was 24 hours. 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.

phase II reactions, based on preliminary assays that included several substrates for glucuronidation, sulfation, and N-acetylation (data not shown; manuscript in preparation). All incubations were performed for 24 hours at 10 mM substrate. Results in skin explants from donor 10 showed that the activity rate of carbazeran 4-hydroxylation was comparable to rates of triclosan glucuronidation and sulfation but ;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

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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. Discussion Although AO has long been known to oxidize nitrogen-containing heterocycles (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, 1985). Based on analysis of chemical structure, a recent review suggested that a large number of drugs on the market (;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 have focused exclusively on the human liver (Obach et al., 2004; Hutzler et al., 2012, 2014), even if gene expression (Nishimura and Naito, 2006) and immunohistochemistry (Moriwaki

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Fig. 4. Time course of carbazeran 4-hydroxylation (A) and zoniporide 2-hydroxylation (B) in the full-thickness human skin explants. Assays were 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 skin punches (squares) and corresponding incubation medium (triangles).

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 (Zientek et al., 2010; Hutzler et al., 2012). 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. Because 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, 1985) and zoniporide 2-hydroxylation (Dalvie et al., 2010, 2012, 2013) (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 (Lebonvallet et al., 2010). Because AO 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 ;10-fold higher relative to zoniporide, with apparent substrate turnover reaching almost 20% for some donors (Fig. 2B; 10 mM incubations for 24 hours). 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 AO 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 the skin of mice, 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 the 4.25-fold difference reported in a set of cryopreserved hepatocytes from 5 donors (Sahi et al., 2008), but considerably lower than the 17-fold variation recently observed for O6-benzylguanine hydroxylation in cryopreserved hepatocytes of 75 donors (Hutzler et al., 2014) or the 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 the human AOX1 gene (Hartmann et al., 2012). The relatively robust activity of AO in the human skin, despite the differences in donor demographics and anatomic region of the skin, may be related to the physiologic role of AO in the metabolism of endogenous retinoids (Tomita et al., 1993; Graessler and Fischer, 2007; Terao et al., 2009). A recent study in AO homolog 2–/– knockout mice observed thickening of the epidermis in basal conditions and after UV light exposure, probably

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Aldehyde Oxidase Activity in Human Skin Explants

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 CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4/5 at concentrations up to 50 mM (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 an estimated concentration to cause 50% cytotoxicity in rat hepatocytes of 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 mM inhibitor (Fig. 3). 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 additional evidence for cutaneous activity of AO.

Reaction rates of carbazeran and zoniporide hydroxylation in skin explants decelerated after 4 hours, suggesting that activities for 24-hour incubations, expressed as picomoles of formed product per milligram of skin tissue per hour, are underestimated (Fig. 4). Although skin histology showed normal microscopic morphology after 24 hours, deviation from linearity is expected due to substrate consumption, metabolite accumulation, or both. For example, Kaye et al. (1985) found an inhibitory effect of 4-hydroxycarbazeran on carbazeran hydroxylation in liver cytosol. Interestingly, the predominant extracellular localization of metabolites after 24 hours 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. Rates of cutaneous carbazeran and zoniporide hydroxylation increased hyperbolically with increasing substrate concentrations (Fig. 5). Apparent substrate affinities (Km,app), as derived from the MichaelisMenten model, were independent of the incubation time, but limiting reaction velocities (Vmax,app) were significantly higher for the 4-hour incubations (Table 3), also reflecting the results of the time-course assay

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 hours. Activity data were fitted to Michaelis-Menten model. Results are presented as the best fit value 6 S.E.M. Carbazeran 4-Hydroxylation

Zoniporide 2-Hydroxylation

Incubation Time Km,app h

mM

24 4

3.57 6 0.48 3.48 6 0.56

Vmax,app –1

r

2

Donor

–1

pmol×mg ×h

1.62 6 0.06 3.46 6 0.16

Km,app mM

0.97 0.95

8 13

20.71 6 4.67 21.37 6 5.75

Vmax,app –1

r2

Donor

0.96 0.95

8 13

–1

pmol×mg ×h

0.37 6 0.04 2.22 6 0.33

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Fig. 5. Enzyme kinetics of carbazeran 4-hydroxylation (A1 and A2) and zoniporide 2-hydroxylation (B1 and B2) in the fullthickness human skin explants. The 24-hour incubations (A1 and B1) were performed with skin from donor 8, whereas 4-hour incubations were performed with skin from donor 11. Activities are presented as the mean value and S.D. Activity data were fitted to MichaelisMenten model. Calculated enzyme kinetic parameters are presented in Table 3.

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Manevski et al. 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, as well as to estimate the contribution of cutaneous AO activity to total metabolic clearance of drugs predominantly eliminated by AO. Acknowledgments The authors thank Marie-Catherine Stutz, Karine Bigot, Arno Doelemeyer, and Armelle Grevot for their help with skin histology and Bertrand-Luc Birlinger, Judith Streckfuss, and Maxime Garnier for providing reagents and technical assistance.

References Fig. 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.

(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 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 mM (Beedham et al., 1987), whereas Dalvie et al. (2010) reported a Km of 3.4 mM for zoniporide 2-hydroxylation in pooled human liver cytosol. Data from existing literature suggest that cutaneous phase II metabolic enzymes, for example, UGTs, SULTs, and NATs (Luu-The et al., 2009; Bonifas et al., 2010a,b; Hu et al., 2010; Jäckh et al., 2011; Kushida et al., 2011; Götz et al., 2012b; van Eijl et al., 2012), have higher expression and activity levels compared with corresponding cutaneous phase I metabolic enzymes (Jäckh et al., 2011; Götz et al., 2012a). 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 (Götz et al., 2012b), together with hydroxylations of carbazeran and zoniporide. As shown in Fig. 6, the activity of carbazeran hydroxylation was comparable to that of triclosan glucuronidation and sulfation and ;3-fold lower than that of 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

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Authorship Contributions Participated in research design: Manevski, Litherland, Schiller, Balavenkatraman. Conducted experiments: Manevski, Bertschi, Ling. Contributed new reagents or analytic tools: Manevski, Bertschi. Performed data analysis: Manevski. Wrote or contributed to the writing of the manuscript: Manevski, Litherland, Balavenkatraman, Ling, Kretz, Pognan, Schiller, Camenisch, Walles, Swart, Wettstein, Schaefer, Wolf.

Aldehyde Oxidase Activity in Human Skin Explants

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Address correspondence to: Dr. Karine Litherland, Novartis Institutes for BioMedical Research, Translational Sciences/DMPK/IDD (Integrated Drug Disposition), Fabrikstrasse 14-1.02.7, Novartis Pharma AG, CH-4002 Basel, Switzerland. E-mail: [email protected]

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