Original Paper Received: December 21, 2012 Accepted after revision: April 25, 2013 Published online: March 15, 2014
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
Effect of Skin Metabolism on Dermal Delivery of Testosterone: Qualitative Assessment using a New Short-Term Skin Model C. Jacques a, c E. Perdu a, b E.L. Jamin a, b J.P. Cravedi a, b A. Mavon c H. Duplan c D. Zalko a, b
a
INRA, and b INP, ENVT, AIPS, UPS, UMR1331, Toxalim (Research Center in Food Toxicology), Université de Toulouse, and c Laboratoire de Pharmacotissulaire et de Pharmacocinétique Cutanée, Pierre Fabre Dermo-cosmétique, Toulouse, France
Key Words Alternative model · Cytochrome P450 · Dermal absorption · Pig skin · Transdermal drug delivery · Testosterone metabolism
Abstract The skin is a metabolically active organ expressing biotransformation enzymes able to metabolize both endogenous molecules and xenobiotics. We investigated the impact of metabolism on the delivery of testosterone through the skin with an ex vivo pig ear skin system as an alternative model for human skin. Penetration, absorption and metabolic capabilities were investigated up to 72 h after application of [14C]-testosterone doses of 50–800 nmol on either fresh or frozen skin, with the latter model being metabolically inactive. Testosterone absorption and metabolite production were monitored by radio-HPLC and gas chromatographymass spectrometry. Testosterone absorption through frozen skin was much lower, irrespective of the dose of testosterone applied, compared to fresh skin. Using fresh skin samples, >95% of the radioactivity recovered in culture media, as well as the skin itself, corresponded to metabolites. These results were compared with the metabolic data obtained from other in vitro systems (liver and skin microsomes). The present work leads to the conclusion that most of the enzymatic activities expressed in liver fractions are also expressed in pig
© 2014 S. Karger AG, Basel 1660–5527/14/0274–0188$39.50/0 E-Mail [email protected] www.karger.com/spp
and human skin. The metabolic activity of the skin can modulate the biological activity of pharmaceuticals (and xenobiotics). Consequently, it can also greatly affect transdermal drug delivery. © 2014 S. Karger AG, Basel
Introduction
Testosterone is the major circulating androgen in man. The biological effects of androgens are numerous, and testosterone deficiency is associated with a number of clinical abnormalities. A variety of preparations containing testosterone, such as oral tablets, intramuscular injections, subcutaneous implants and transdermal application, is available as substitution therapy of climacteric symptoms as well as hypogonadism [1, 2]. Among the several possible administration routes for testosterone, transdermal delivery offers advantages over oral administration, in that hepatic first-pass metabolism in the liver occurs for the oral route, which requires higher testosterone doses. Cutaneous absorption of different formulation types of testosterone has been well documented over the last decade. The relatively low molecular weight of 288 and moderate lipophilicity (log Kow = 3.3) of testosterone are factors favoring transdermal delivery [3]. Carine Jacques INRA, UMR1331, Toxalim Research Center in Food Toxicology 180, chemin de Tournefeuille FR–31027 Toulouse (France) E-Mail carine.jacques @ pierre-fabre.com
The penetration of chemicals through the stratum corneum is generally considered to be the key step that limits percutaneous absorption, thereby dictating the systemic bioavailability of topically applied molecules. However, the skin is a metabolically active tissue expressing biotransformation enzymes. These enzymes are able to metabolize xenobiotics which are in contact with skin [4]. These metabolic processes can play an important role in the fate of topically applied substances and therefore modulate their systemic bioavailability [5, 6]. The ability of the skin to activate or detoxify xenobiotics has been extensively demonstrated in in vitro studies [7]. The skin expresses functional phase I oxidative, reductive and hydrolytic reactions, as well as phase II conjugation capabilities [8–11]. Testosterone is known to be metabolized through CYP450-dependent pathways into several reduced and oxidized metabolites. It is well recognized that steroid metabolism is particularly active in the pilosebaceous unit, and that keratinocytes extensively metabolize steroids [12–14]. However, little information detailing the cutaneous biotransformation of testosterone and its possible impact on transdermal testosterone delivery has been published previously. Percutaneous absorption and metabolism studies are essential to gain a sound understanding of the toxicity of compounds that can come in contact with the skin surface. They are also of prime importance in the development of drugs for dermal or transdermal application. Due to the limited accessibility of viable human skin explants, commercially available reconstructed human epidermis models (skin equivalents) are evaluated as an alternative. For topically applied molecules, one of the main limits of these models is their weak barrier function [15], which can lead to an overestimation of diffusion and biotransformation rates [16]. In this context, we used a short-term skin culture model, which we previously developed using skin explants from domestic pig ears purchased at the slaughterhouse [17–19], to examine the capability of the skin to biotransform topically applied testosterone and investigate the influence of testosterone biotransformation on its delivery. In order to standardize the predictive testing of chemicals and cosmetics for regulatory purposes, the Organization for Economic Cooperation and Development (OECD) adopted guideline 28 and a corresponding technical guidance document to describe methods assessing drug absorption using human and animal skin ex vivo [20]. In this guideline, testosterone is clearly suggested as one of the model molecules to develop a skin alternative model. As part of the validation process of the pig ear short-term skin culture model, we Cutaneous Testosterone Metabolism
used radiolabeled testosterone applied on fresh versus previously frozen skin explants. In the latter model, biotransformation capabilities have been inactivated by the freezing process (2 months), but the skin barrier function remains intact [5]. The delivery of radiolabeled testosterone was followed for a time period of 72 h. Testosterone and its metabolites were quantified at 24, 48 and 72 h using HPLC coupled to online radioactivity detection. The metabolite structure was investigated by mass spectrometry (MS). A concentration range of 50–800 nmol (6 different doses of testosterone) was used to study the metabolic capability of the pig ear skin model and to examine the viability of the model over a 72-hour time period. These metabolic data were then compared with the results obtained from incubations of testosterone with human and pig liver as well as skin microsomes.
Materials and Methods Chemicals [4,7-14C]-testosterone with a specific activity of 2.03 GBq/ mmol was purchased from Amersham Biosciences (Little Chalfont, UK). Its radiochemical purity was >97.5% based on radioHPLC analysis. Unlabeled testosterone, purchased from SigmaAldrich (St-Quentin-Fallavier, France) was >99% pure. The following reference metabolites were purchased from Sigma-Aldrich: 2β-hydroxytestosterone (2β-OH-Testo), 6β-hydroxytestosterone (6β-OH-Testo), 7α-hydroxytestosterone (7α-OH-Testo), 11βhydroxytestosterone (11β-OH-Testo), 16α-hydroxytestosterone (16α-OH-Testo), 11-ketotestosterone (11-keto-Testo), 1-dehydrotestosterone (Δ1-Testo), 4-androstene-3,17-dione (Δ4-dione), 4-androsten-11β-ol-3,17-dione (11β-OH-Δ4-dione), 4-androsten16α-ol-3,17-dione (16α-OH-Δ4-dione), epitestosterone (epiTesto), androsterone, epiandrosterone, 5β-androstan-3α-ol-17-one (5β-A-3α-17-one), 5β-androstan-3β-ol-17-one (5β-A-3β-17one), 5α-androstan-3,17-dione (5α-A-dione) and 5β-androstan3,17-dione (5β-A-dione). Other chemicals and solvents (of analytical grade) were purchased from the following sources: ammonium acetate, sodium acetate, phosphate buffer (0.01 M) and sodium hydroxide from Sigma-Aldrich; acetonitrile and ethyl acetate from Scharlau Chemie S.A. (Barcelona, Spain), and ethanol and acetic acid from Merck (Briare-Le-Canal, France). Ultrapure water, obtained using a Milli-Q system (Millipore, Saint-Quentin-en-Yvelines, France), was used for ex vivo preparations and for the preparation of HPLC mobile phases. Dulbecco’s modified Eagle’s medium and L-glutamine were obtained from GibcoTM (Cergy-Pontoise, France). Antibiotics (streptomycin/penicillin and gentamycin) and fungizone, used in the culture media, were purchased from Sigma-Aldrich. Pig Ear Skin Short-Term Cultures Fresh Skin The preparation and incubation conditions of pig skin explants have been previously detailed by Jacques et al. [17]. Briefly, skin cultures were developed from ears of domestic pigs (Pietrain
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breed, 6-month-old females) as these were easily obtained from a local slaughterhouse. After cleaning and shaving, the skin was immediately excised with a scalpel and sectioned at a thickness of 500 μm and punched in 28-mm-diameter discs. Skin punches were placed dermal side down in 23-mm-diameter inserts in 6-well plates prefilled with 1.5 ml of culture medium per well at 37 ° C in a CO2 air incubator. [14C]-testosterone (8,333 Bq/sample), which was adjusted with unlabeled testosterone to reach the required doses of 50, 100, 200, 400, 600 and 800 nmol (corresponding to 3.03, 6.06, 12.12, 24.25, 36.39 and 48.52 μg/cm2, respectively), was diluted in 60 μl of phosphate buffer/ethanol (1:1, v/v) and applied to the skin surface. Culture media collected at 24 and 48 h and at the end of the experiment (72 h) were stored at –20 ° C until analysis. All incubations were carried out in triplicate.
Frozen Skin The influence of skin viability on percutaneous absorption was investigated by comparing testosterone diffusion in fresh explants and in identical skin preparations which had previously been frozen (2 months at –20 ° C). After this enzyme inactivation period, skin punches were incubated similarly as fresh skin with 50, 200 and 800 nmol [14C]-testosterone (8,333 Bq) applied to the skin punches, respectively.
Compartmental Analysis of Radiolabeled Testosterone Compartmental analysis was carried out as previously described [17–19]. Briefly, skin surfaces, tissue culture inserts and wells were washed twice with methanol. Testosterone and metabolites were extracted from the skin after homogenization with a Polytron homogenizer (Kinematica, Luzern, Switzerland) in methanol/phosphate buffer at pH 7.4 (2: 1; v/v) and centrifuged. Radioactivity in culture media, skin extracts and washing solutions was quantified using a scintillation counter (Tri-Carb; Perkin-Elmer, Waltham, Mass., USA). Microsomal Fractions Liver microsomes obtained from pools of 10 human male and female donors were purchased from TEBU (Le-Perray-en-Yvelines, France). Human skin microsomes (pool of 2 donors) were purchased from Biopredic (Rennes, France). Pig liver and skin microsomes were prepared from domestic pigs as previously described by Jacques et al. [17, 18] and Zalko et al. [19]. All microsomes were stored at –80 ° C until use. The protein content of microsomal preparations was determined according to the method of Lowry et al. [21].
Metabolism of Testosterone by Skin and Liver Microsomes Radiolabeled [14C]-testosterone (3,333 Bq) fortified with unlabeled testosterone to reach the required concentrations (1, 5, 10, 50, 100, 150 and 200 μM, respectively) was incubated for 20 min at 37 ° C under shaking with 2 mg of microsomal protein from human liver or pig liver, respectively. Similar conditions were used for experiments carried out using human and pig skin microsomes with two testosterone concentrations (1 and 5 μM). Incubations were performed in a final volume of 1 ml of 0.1 M sodium phosphate buffer, 5 mM MgCl2 (pH 7.4) containing an NADPH-generating system (1.3 mM NADP, 5 mM glucose-6-phosphate and 2 IU glucose-6-phosphate dehydrogenase). All pig microsome incubations were performed in triplicate. Blank samples without an NADPH
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generating system were used as controls for non-NADPH-mediated testosterone transformations. At the end of the incubation period, the reaction was stopped by protein precipitation with the addition of 3 vol methanol and centrifugation for 10 min at 8,000 g. Supernatant aliquots (500 μl) were concentrated under a gentle nitrogen stream and analyzed by HPLC coupled to online radioactivity detection. Metabolite identification was performed by MS analysis. Analysis of Testosterone by Radio-HPLC For all samples (culture media, skin extracts and microsomal incubations), 3 vol methanol were added. Samples were stirred for 1 min and then centrifuged. Supernatants were concentrated under a gentle nitrogen stream and metabolites were separated by radio-HPLC. The HPLC system consisted of a Spectra P1000 pump (Thermo Separation Products, Les Ulis, France) associated with a 250 × 4 mm (5-μm) Kromasil-C18 column (Bischoff, Montluçon, France) protected by a Kromasil-C18 guard column (Interchim, Montluçon, France). Mobile phases used at a flow rate of 1 ml/min consisted of ammonium acetate (20 mM, pH 3.5) and acetonitrile (A). The temperature of the column was maintained at 35 ° C and the following gradients were used: 0 min, 20% A; 35 min, 40% A; 50 min, 60% A; 55 min, 100% A; 60 min, 20% A. [14C]-testosterone and related metabolites were monitored by online radioactivity detection using a Flo-one A500 (Radiomatic, LaQueue-Lez-Yvelines, France) and Flow-scint II (Packard Instruments, Downers Grove, Ill., USA) as the scintillation cocktail. Testosterone and its metabolites were quantified by integrating the area of the radiochromatographic peaks. The limit of detection is 20 dpm and corresponds to three times the noise signal on the radiochromatogram and the limit of quantitation is 70 dpm and corresponds to ten times the noise signal on the radiochromatogram.
Structural Characterization of Testosterone Metabolites Metabolites were isolated using HPLC separation coupled to metabolite collection using a Gilson model 201/202 fraction collector (Gilson France, Villiers-Le-Bel, France). Radioactivity in the different fractions was determined by direct counting of sample aliquots on the Packard scintillation counter. Fractions of interest were pooled and dried under a gentle nitrogen stream for MS analyses. For the less water-soluble metabolites (typically those with a retention time above 30 min in HPLC), no derivatization step was required prior to gas chromatography (GC) MS analysis. Such samples, once isolated, were directly dissolved in 40 μl of methanol before injection. For the other metabolites, including OH-Testo and OH-Δ4-dione, a derivatization step was required prior to GCMS, to ensure an efficient detection. For this purpose, 50 μl of N,Obis(trimethylsilyl)trifluoroacetamide supplemented with 1% trimethylchlorosilane (Grace Davison Discovery Sciences, Deerfield, Ill., USA) were added to the samples. The derivatization reaction proceeded for 12 h at 60 ° C. Then, reaction mixtures were evaporated to dryness under a nitrogen stream and immediately dissolved with 40 μl of hexane. GC-MS analyses were performed on a Trace GC 2000 device associated with a Polaris ion trap mass analyzer (Thermo Scientific, Les Ulis, France). The injection volume was 2 μl in the splitless mode at 270 ° C. Chromatographic separation was achieved on an Optima 5 Accent capillary column (30 m × 0.25 μm, 0.25 μm) from Macherey-Nagel (Hoerdt, France). Helium was used as the carrier
Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
Diffusion of testosterone and its metabolites (nmol/cm2)
Fig. 1. Diffusion of testosterone in culture
90 72 h 48 h 24 h
80 70 60 50 40 30 20 10 0
50
100 200 400 600 Dose of testosterone applied on skin explants (nmol)
media as a function of the incubation time for different doses (means ± SD, n = 3).
800
Table 1. Radioactivity distribution in media, skin and washing solutions 72 h after the beginning of the experi-
ment for each testosterone dose applied on the skin surface Testosterone dose 50 nmol
100 nmol
200 nmol
400 nmol
600 nmol
800 nmol
Skin surface Skin Culture media Culture insert Well washing
0.7±0.4 9.9±0.8 84.2±15.7 0.6±0.1 3.0±0.2
0.6±0.1 16.1±5.2 86.5±4.6 0.9±0.2 3.6±0.8
0.7±0.1 19.0±6.8 80.5±4.0 0.9±0.1 3.8±0.8
1.0±0.2 25.1±5.8 72.5±3.5 1.1±0.3 3.4±1.1
1.5±0.4 32.0±1.7 59.0±6.2 1.1±0.2 3.3±1.1
1.9±2.3 32.7±1.9 41.3±9.6 1.7±1.6 1.6±0.4
Total
98.5±16.6
107.4±10.6
104.9±12.1
103.3±9.0
97.4±8.2
79.3±12.7
Results are expressed as percent of the applied radioactivity (means ± SD, n = 3).
gas at a flow rate of 1 ml/min. The following oven program was used: 40 ° C for 1 min, then 40–250 ° at 25 ° C/min (8.4 min), 250– 280 ° at 5 ° C/min (6 min) and a final hold time of 10 min at 280 ° C. The injector and GC-MS interface were set at 250 ° C. Positive electron ionization (EI) mass spectra (70 eV) were generated at a source temperature of 220 ° C. Spectra were acquired from m/z 60–550.
Results
Distribution of Testosterone in Pig Ear Skin: Short-Term Cultures Table 1 summarizes the compartmental distribution of radioactivity (expressed as percent testosterone equivalent) at 72 h. Mass balance recovery for 50–600 nmol testosterone ranged between 97.4 and 107.4%. The mass balance met the requirements of the Scientific Committee Cutaneous Testosterone Metabolism
on Consumer Safety guidelines. Only at the highest dose (800 nmol), mass balance recovery dropped slightly below 85%. The radioactivity remaining on the surface of the skin at the end of the experiments (72 h) ranged between 0.6 and 1.9% of the applied radioactivity. In the skin itself, radioactivity levels gradually increased from 9.9 (50 nmol testosterone) to 32.7% (800 nmol testosterone). Conversely, the main part of the radioactivity was recovered in culture media, but with decreasing percentages for testosterone doses of 50 (84.2%) to 800 nmol (41.3%). Residual radioactivity recovered from inserts and wells never exceed (together) 4.7% of the applied doses. Figure 1 details the diffusion of testosterone in culture media as a function of the incubation time (24, 48 and 72 h) and for the different doses tested. Whatever the apSkin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
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100
Testosterone Metabolites
90
Radioactivity (%)
80 70 60 50 40 30 20 10 0
50
a
200
800
50
Surface skin
200
800
50
Skin
200
800
Medium
90 Testosterone Metabolites
80
Radioactivity (%)
70 60 50 40 30 20 10
Fig. 2. Comparison of radioactivity distri-
bution (testosterone + metabolites, 72 h) in fresh (a) and frozen skin explants (b). Results are expressed as percent of the applied radioactivity (mean ± SD, n = 3).
0
b
50
200 Surface skin
plied dose, the flux of testosterone (sum of the parent compound and metabolites, based on culture medium counts) decreased during each successive 24-hour period. For instance, when applying 600 nmol of testosterone, 40.1, 24.5 and 20.5 nmol of testosterone equivalent/cm2 were recovered in the culture medium following the three 24-hour time periods, respectively. At this concentration, testosterone flux was 1.67, 1.02 and 0.99 nmol/h/cm2, respectively. This decrease in testosterone flux was likely due to the progressive depletion of available testosterone, and this trend was confirmed for all the other concentrations tested. Fresh versus Frozen Skin Figure 2 presents the compartmental analysis of 3 different doses of testosterone and its metabolites for fresh (fig. 2a) and skin frozen for 2 months prior to the experi192
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
800
50
200 Skin
800
50
200
800
Medium
ments (fig. 2b). For frozen skin, the mass balance was found to meet OECD guidelines, but the quantity of radioactivity remaining at the surface of the skin at 72 h was slightly higher than in experiments carried out with fresh skin. In the skin itself, the percentage of radioactivity was also higher for frozen skin. Conversely, the diffusion of testosterone within fresh skin, estimated by the percentage of radioactivity in culture media, was higher than in frozen skin whatever the dose of testosterone applied. For instance, in 50-nmol assays, 84.2 ± 5.1 and 75.4 ± 3.8% of the applied dose was recovered in culture media of fresh and frozen skin, respectively. Culture media as well as skin extracts were analyzed by radio-HPLC (fig. 3). When frozen skin was used, the radioactivity recovered in culture media and skin corresponded to the parent testosterone only, indicating Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
2,750
Testosterone
2,500 2,250
DPM
2,000 1,750 1,500 1,250 1,000 750 500 250 0
0
10
20
30
a
40
50
60
Time (min) 1,000
VIII
900 800 700
XIII
DPM
600 XI
500
XII
400
XIII’
300 Testosterone
200 100
Fig. 3. Typical radiochromatographic pro-
files obtained from culture media using frozen (a) and fresh skin explants (b) exposed to [14C]-testosterone (200 nmol, 72 h).
0
b
I
0
5
II
10
III
IV V’
15
X
VI IX’
VII
20
25
30 35 Time (min)
40
45
50
55
60
65
that no biotransformation had taken place. Conversely, when using fresh skin explants, a high proportion of the radioactivity, both in culture media and skin extracts, corresponded to testosterone metabolites, which eluted either before or after testosterone under our HPLC conditions. A representative radiochromatogram displayed in figure 3 corresponds to the analyses of 72hour samples of culture media, frozen (fig. 3a) and fresh (fig. 3b) skin explants, respectively, for incubations carried out with 200 nmol of testosterone. Fourteen peaks were detected in the culture media. All were also detected in skin extracts. At the end of the experiment,
metabolites in culture media ranged from 81.4 [50-nmol assays] to 39.9% [800-nmol assays] of the dose, while the proportion of metabolites in skin extracts gradually increased from 9.9 [50-nmol assays] to 32.6% [800-nmol assays]. For the 50-nmol dose, peak VIII was the major metabolite detected in culture medium samples (75.9 ± 4.7%) as well as skin extracts (88.1 ± 1.6%). Other metabolites detected in culture media and in skin extracts accounted, all together, for 22.4 ± 4.9 and 13.1 ± 3.1% of the radioactive dose, respectively. Only low rates of unmetabolized testosterone were recovered in culture me-
Cutaneous Testosterone Metabolism
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Table 2. GC-MS (EI) identification of testosterone metabolites formed in incubations of testosterone carried out with liver microsomes or pig ear skin explants (for which both culture media and skin extracts were analyzed)
Peak No.
Metabolite
Radio-HPLC min
GC RT min
Standard, GC RT min
M+· ions m/z
I II III IV Vb V’c VI VII VIII IXb IX’c X Testo XI XII XIII’b XIII
7α-OH-Testo 6β-OH-Testo 16α-OH-Testo OH-Δ4-dione OH-Testo OH-Δ4-dione 16α-OH-Δ4-dione OH-Testo OH-Δ4-dione OH-Testo OH-Δ4-dione Δ6-Testo testosterone androstenedione epiandrosterone 5α-A-dione androsterone
9.0 12.0 14.0 16.0 18.0 17.5 20.0 21.0 25.5 28.0 27.9 33.0 35.4 40.0 43.0 47.0 48.0
14.20 14.23 15.79 14.92 15.70 15.70 15.16 14.49 15.67 15.81 15.79 14.61 14.30 14.14 13.30 13.59 13.24
7α-OH-Testo-TMS2 (14.20) 6β-OH-Testo-TMS2 (14.23) 16α-OH-Testo-TMS2 (15.79) standard not available standard not available standard not available 16α-OH-Δ4-dione-TMS (15.16) standard not available standard not available standard not available standard not available standard not availabled testosterone (14.30) androstenedione (14.14) epiandrosterone (13.30) 5α-A-dione (13.60) androsterone (13.24)
448.0a 448.0a 448.0a 374.0a 448.0a 374.0a 374.0a 448.0a 374.0a 448.0a 374.0a 286.1 288.2 286.1 290.2 288.1 290.1
a Derivatized
with N,O-bis(trimethylsilyl)trifluoroacetamide-trimethylchlorosilane (1%). observed in pig ear skin explants. c Only observed in microsomal incubations. d Identification made on the basis of Brown and Djerassi [22] in 1981. RT = Retention time. b Only
dia as well as skin extracts, irrespective of the dose applied. These results demonstrated extensive biotransformation of the model substrate. Structural Characterization of Skin Testosterone Metabolism Metabolism of Testosterone by Short-Term Skin Cultures: Qualitative Analysis Metabolites collected from culture media and skin extracts were analyzed by GC-MS using EI ionization. GCMS data are summarized in table 2. The most polar metabolites, which correspond to compounds eluted before 30 min, did require a derivatization step prior to GC-MS analysis. Trimethylsilyl derivates (TMS) of hydroxylated metabolites produced characteristic losses of a methyl radical and HOSi(CH3)3 from M+ · ions in EI MS. Accordingly, peaks I, II, III, V, VII and IX were all identified as testosterone hydroxylated metabolites (OH-Testo), since their respective mass spectra displayed two derivatized hydroxyl functions, as well as fragment ions in full concordance with such structure (data not shown). Analyses performed with standard testosterone metabolites by GC-MS further allowed to detail the structural character
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ization of these metabolites. Thus, peaks I, II and III were attributed to 7α-OH-Testo, 6β-OH-Testo and 16α-OHTesto, respectively. The position of the hydroxyl group of other OH-Testo metabolites could not be determined on the basis of the comparison with available standards. Metabolites corresponding to peaks IV, VI and VIII were identified as hydroxyandrostenediones (OH-Δ4-diones), since their respective mass spectra showed one derivatized function and a fragmentation pattern consistent with a OH-Δ4-dione structure. Peak VI was identified as the 16α-Δ4-dione following comparison with the corresponding authentic standard. The hydroxylation position of other OH-Δ4-dione metabolites could not be determined on the basis of standard analyses. Alternatively, metabolites eluted after 30 min were submitted directly to GC-MS analyses (i.e. without TMS derivatization). The compound which eluted at 35.4 min in radio-HPLC was confirmed to be testosterone. The mass spectrum of peak X displayed M+ · ions at m/z 286 with a characteristic fragmentation pattern of 17β-hydroxyandrosta-4,6-dien-3-one (Δ6-Testo) and diagnostic fragment ions detected at m/z 136 [22]. The mass spectrum and GC retention time of peak XI were both
Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
Metabolites formed (nmol/h)
3.0 2.5
2+©GLRQH Epiandrosterone Androstenedione Androsterone į$GLRQH ©7HVWR į2+7HVWR DŽ2+7HVWR į2+7HVWR į©GLRQH
2.0 1.5 1.0 0.5 0 0
100
200
300
400
500
600
700
800
Testosterone dose applied on skin (nmol)
Fig. 4. Testosterone metabolite production at 72 h as a function of the testosterone dose applied on skin explants. Calculations are based on concentrations measured in culture media and skin extracts (means ± SD, n = 3).
consistent with the results obtained for the androstenedione standard. Similarly, GC-MS analyses of peaks XII, XIII and XIV were compared with the analyses of the corresponding standards, allowing the attribution of these metabolites to epiandrosterone, 5α-A-dione and androsterone, respectively, as shown in table 2. Metabolism of Testosterone by Skin Short-Term Cultures: Quantitative Analysis Production rates (in nmol/h) of testosterone metabolites in skin short-term cultures were calculated based on radio-HPLC analyses. Kinetic curves were plotted as a function of the testosterone dose (fig. 4). Peak VIII (OH-Δ4-dione) was the major metabolite produced by skin short-term cultures. For this metabolite, a saturation occurring at around 400 nmol of testosterone was observed and followed Michaelis-Menten kinetics, with Vm and Km values of 3.09 ± 0.33 nmol/h/mg and 165.4 ± 55.4 μM, respectively. The formation rate of all other metabolites (epiandrosterone, androsterone, androstenedione, 5α-A-dione, OH-Testo, 6β-OH-Testo, 7α-OH-Testo, 16α-OH-Testo, 16α-Δ4-dione and OHΔ4-diones) also followed Michaelis-Menten kinetics, and, as observed for OH-Δ4-dione, seemed to reach saturation around a testosterone dose of 400–800 nmol. However, for these metabolites, the range of concentrations selected for the study did not allow a precise determination of the saturating dose and thus the calculation of kinetic constants. Cutaneous Testosterone Metabolism
Testosterone Metabolism by Liver and Skin Microsomes of Pig and Human Origin Metabolism of Testosterone by Liver and Skin Microsomes: Qualitative Analysis In incubations of [14C]-testosterone with microsomes (either from pig liver, pig skin, human liver or human skin), always >90% of the radioactivity put in incubations was recovered at the end of the experiments. In our analytical conditions, the retention time of testosterone was 35.4 min. Qualitatively, similar metabolic profiles were observed for incubations carried out with human or pig microsomal fractions. Using liver microsomes, a large proportion of the radioactivity corresponded to testosterone metabolites >54.5%. The radio-HPLC profiles obtained for these incubations were also very similar to the radio-HPLC profile obtained for fresh pig ear skin explants. For skin microsome incubations, only three groups of metabolites were detected (peaks III, XI and XII). Finally, none of the metabolites was observed when no NADPH-generating system was added to the incubations (data not shown), demonstrating that their formation was cytochrome P450 dependent. Structural Characterization of Testosterone Metabolites Some metabolites displayed identical HPLC retention times as those produced by skin culture (peaks I–IV, VI– VIII, X–XII). All GC-MS results are summarized in table 2. Metabolites eluting before 30 min in our radioSkin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
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HPLC system (peaks I–IX’) required to be derivatizated prior to their characterization by GC-MS. Metabolites corresponding to peaks I, II, III and VII were identified as OH-Testo, similarly to the corresponding metabolites produced by pig skin. Comparison with GC-MS analyses of standard metabolites allowed to identify peaks I, II and III as 7α-OH-Testo, 6β-OH-Testo and 16α-OH-Testo, respectively. The hydroxylation position of other OHtestosterone metabolites was not determined on the basis of available standards. OH-Testo metabolites V and IX produced by skin short-term culture were not detected in microsomal incubations. Conversely, OH-Δ4-dione metabolites were characterized (peaks V’ and IX’). Peak VI was confirmed to be 16α-Δ4-dione. We were unable to determine the position of the hydroxyl group of other OH-Δ4-dione metabolites. Metabolites eluted with a retention time >30 min were directly analyzed by GC-MS. Retention times and mass spectra allowed to attribute peaks X and XI to Δ6-Testo and androstenedione, respectively. Similarly, GC-MS analyses of peaks XII and XIV compared with analyses of appropriate standards permitted to identify these metabolites as epiandrosterone and androsterone, respectively (table 2). Finally, metabolite XIII was only observed in pig ear skin samples and was identified as 5α-A-dione by GCMS analysis. Radio-HPLC and GC-MS analyses of incubations with liver microsomes showed that the same metabolites were generated in pig and human samples (table 2). Due to the low amount of testosterone metabolites formed by pig and human skin microsomes, their identification was based only on HPLC retention times comparison with authentic standards and/or previously identified biotransformation products. For both species, the three groups of metabolites detected by radio-HPLC had retention times which were similar to the three of the metabolites produced by liver microsomes (table 2), respectively, namely 16α-OH-Testo (peak III), androstenedione (peak XI) and epiandrosterone (peak XII). Metabolism of Testosterone by Liver and Skin Microsomes: Quantitative Analysis Testosterone metabolic rates were investigated over a substrate concentration range from 1 to 200 μM for pig liver microsomes. Human liver and skin microsomes were incubated with two testosterone concentrations (1 and 5 μM). The main metabolites formed by liver and skin microsomes from humans and pigs are represented in the figure 5. For humans as well as pigs, overall testosterone biotransformation rate was lower in the skin com196
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
pared to the liver. For example, at a substrate concentration of 5 μM, 2.73 ± 0.88 nmol of testosterone metabolites were formed in pig liver microsomal incubations, whereas only 0.26 ± 0.08 nmol were produced with pig skin microsomes. The metabolic rate of testosterone by skin microsomes was approximately 10 times lower than that with liver microsomes. Major metabolites detected in liver microsome incubations were also detected in skin microsome incubations (16α-OH-Testo, androstenedione and epiandrosterone). Other metabolites, which were identified in liver microsome incubations, could not be detected in skin microsome incubations, probably due to analytical limits of detection (20 dpm for radio-HPLC).
Discussion
In this work, the absorption and metabolic capabilities of a pig ear skin system were studied to investigate the specific hydroxylation activities expressed in this ex vivo skin model and to examine the impact of testosterone biotransformation on its bioavailability. Testosterone is an endogenous hormone with a complex metabolism and a model compound recommended by the OECD. The low molecular weight (288) and relatively lipophilic nature (log Kow = 3.3) of testosterone are factors that improve skin penetration [23]. Furthermore, the pattern of testosterone hydroxylation is commonly used to characterize phase I metabolism by different isoforms of cytochrome P450 in the rat liver. Several models enabling the study of fate of testosterone are currently available, but there are little data taking biotransformation issues for this molecule into account. The penetration (skin barrier function), absorption and biotransformation capabilities (phase I and II metabolic activities) of the ex vivo pig skin model were investigated using labeled testosterone on fresh (viable) and frozen (metabolically inactivated) skin. Compared to incubations carried out with fresh skin, testosterone absorption seemed to be weaker with frozen skin, but the observed difference was not statistically significant. Conversely, a tendency was observed for the amount of radioactivity remaining at the surface of the skin by the end of the experiments, with higher values recorded for the surface of the skin and in the skin, itself. However, from a qualitative point of view, striking differences were observed between fresh and frozen skin incubations, with only parent testosterone being recovered in culture media for the latter system. Using fresh skin explants, the major part of the radioactivity recovered in culture media as well Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
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as in the skin itself corresponded to metabolites (>95%). The formation of metabolites increased with the concentration of testosterone, and a beginning of saturation was observed only after application of 400 nmol of testosterone or more. Metabolism was shown to follow MichaelisMenten kinetics. These data demonstrate that testosterone was extensively metabolized by the skin, and that the barrier effect of the ex vivo pig ear skin model was preserved over the whole duration of the experiment. This is fully consistent with our previous work on benzo(a)pyrene, with the same ex vivo pig ear skin model [17, 24]. When using viable epidermis, the rate of transport of the parent molecule is often the limiting factor. In the case of
a lipophilic compound, and if the parent molecule is metabolized into a more hydrophilic compound, percutaneous absorption of the metabolite can be greatly enhanced and actually can be faster than the absorption of the parent compound [25]. In our study, it is remarkable that >95% of the testosterone diffused into the skin are in the form of metabolites. Testosterone was biotransformed into androstenedione, 5α-A-dione, androsterone, epiandrosterone, Δ6Testo, OH-Δ4-diones and OH-Testo, which oxidated at different positions of the molecule (fig. 6). Combined with the kinetic parameters detailed in the results section, this demonstrates that oxidative enzymes were function-
Cutaneous Testosterone Metabolism
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
197
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Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
Effect of Skin Metabolism on Dermal Delivery of Testosterone: Qualitative Assessment using a New Short-Term Skin Model C. Jacques a, c E. Perdu a, b E.L. Jamin a, b J.P. Cravedi a, b A. Mavon c H. Duplan c D. Zalko a, b
a
INRA, and b INP, ENVT, AIPS, UPS, UMR1331, Toxalim (Research Center in Food Toxicology), Université de Toulouse, and c Laboratoire de Pharmacotissulaire et de Pharmacocinétique Cutanée, Pierre Fabre Dermo-cosmétique, Toulouse, France
Key Words Alternative model · Cytochrome P450 · Dermal absorption · Pig skin · Transdermal drug delivery · Testosterone metabolism
Abstract The skin is a metabolically active organ expressing biotransformation enzymes able to metabolize both endogenous molecules and xenobiotics. We investigated the impact of metabolism on the delivery of testosterone through the skin with an ex vivo pig ear skin system as an alternative model for human skin. Penetration, absorption and metabolic capabilities were investigated up to 72 h after application of [14C]-testosterone doses of 50–800 nmol on either fresh or frozen skin, with the latter model being metabolically inactive. Testosterone absorption and metabolite production were monitored by radio-HPLC and gas chromatographymass spectrometry. Testosterone absorption through frozen skin was much lower, irrespective of the dose of testosterone applied, compared to fresh skin. Using fresh skin samples, >95% of the radioactivity recovered in culture media, as well as the skin itself, corresponded to metabolites. These results were compared with the metabolic data obtained from other in vitro systems (liver and skin microsomes). The present work leads to the conclusion that most of the enzymatic activities expressed in liver fractions are also expressed in pig
© 2014 S. Karger AG, Basel 1660–5527/14/0274–0188$39.50/0 E-Mail [email protected] www.karger.com/spp
and human skin. The metabolic activity of the skin can modulate the biological activity of pharmaceuticals (and xenobiotics). Consequently, it can also greatly affect transdermal drug delivery. © 2014 S. Karger AG, Basel
Introduction
Testosterone is the major circulating androgen in man. The biological effects of androgens are numerous, and testosterone deficiency is associated with a number of clinical abnormalities. A variety of preparations containing testosterone, such as oral tablets, intramuscular injections, subcutaneous implants and transdermal application, is available as substitution therapy of climacteric symptoms as well as hypogonadism [1, 2]. Among the several possible administration routes for testosterone, transdermal delivery offers advantages over oral administration, in that hepatic first-pass metabolism in the liver occurs for the oral route, which requires higher testosterone doses. Cutaneous absorption of different formulation types of testosterone has been well documented over the last decade. The relatively low molecular weight of 288 and moderate lipophilicity (log Kow = 3.3) of testosterone are factors favoring transdermal delivery [3]. Carine Jacques INRA, UMR1331, Toxalim Research Center in Food Toxicology 180, chemin de Tournefeuille FR–31027 Toulouse (France) E-Mail carine.jacques @ pierre-fabre.com
The penetration of chemicals through the stratum corneum is generally considered to be the key step that limits percutaneous absorption, thereby dictating the systemic bioavailability of topically applied molecules. However, the skin is a metabolically active tissue expressing biotransformation enzymes. These enzymes are able to metabolize xenobiotics which are in contact with skin [4]. These metabolic processes can play an important role in the fate of topically applied substances and therefore modulate their systemic bioavailability [5, 6]. The ability of the skin to activate or detoxify xenobiotics has been extensively demonstrated in in vitro studies [7]. The skin expresses functional phase I oxidative, reductive and hydrolytic reactions, as well as phase II conjugation capabilities [8–11]. Testosterone is known to be metabolized through CYP450-dependent pathways into several reduced and oxidized metabolites. It is well recognized that steroid metabolism is particularly active in the pilosebaceous unit, and that keratinocytes extensively metabolize steroids [12–14]. However, little information detailing the cutaneous biotransformation of testosterone and its possible impact on transdermal testosterone delivery has been published previously. Percutaneous absorption and metabolism studies are essential to gain a sound understanding of the toxicity of compounds that can come in contact with the skin surface. They are also of prime importance in the development of drugs for dermal or transdermal application. Due to the limited accessibility of viable human skin explants, commercially available reconstructed human epidermis models (skin equivalents) are evaluated as an alternative. For topically applied molecules, one of the main limits of these models is their weak barrier function [15], which can lead to an overestimation of diffusion and biotransformation rates [16]. In this context, we used a short-term skin culture model, which we previously developed using skin explants from domestic pig ears purchased at the slaughterhouse [17–19], to examine the capability of the skin to biotransform topically applied testosterone and investigate the influence of testosterone biotransformation on its delivery. In order to standardize the predictive testing of chemicals and cosmetics for regulatory purposes, the Organization for Economic Cooperation and Development (OECD) adopted guideline 28 and a corresponding technical guidance document to describe methods assessing drug absorption using human and animal skin ex vivo [20]. In this guideline, testosterone is clearly suggested as one of the model molecules to develop a skin alternative model. As part of the validation process of the pig ear short-term skin culture model, we Cutaneous Testosterone Metabolism
used radiolabeled testosterone applied on fresh versus previously frozen skin explants. In the latter model, biotransformation capabilities have been inactivated by the freezing process (2 months), but the skin barrier function remains intact [5]. The delivery of radiolabeled testosterone was followed for a time period of 72 h. Testosterone and its metabolites were quantified at 24, 48 and 72 h using HPLC coupled to online radioactivity detection. The metabolite structure was investigated by mass spectrometry (MS). A concentration range of 50–800 nmol (6 different doses of testosterone) was used to study the metabolic capability of the pig ear skin model and to examine the viability of the model over a 72-hour time period. These metabolic data were then compared with the results obtained from incubations of testosterone with human and pig liver as well as skin microsomes.
Materials and Methods Chemicals [4,7-14C]-testosterone with a specific activity of 2.03 GBq/ mmol was purchased from Amersham Biosciences (Little Chalfont, UK). Its radiochemical purity was >97.5% based on radioHPLC analysis. Unlabeled testosterone, purchased from SigmaAldrich (St-Quentin-Fallavier, France) was >99% pure. The following reference metabolites were purchased from Sigma-Aldrich: 2β-hydroxytestosterone (2β-OH-Testo), 6β-hydroxytestosterone (6β-OH-Testo), 7α-hydroxytestosterone (7α-OH-Testo), 11βhydroxytestosterone (11β-OH-Testo), 16α-hydroxytestosterone (16α-OH-Testo), 11-ketotestosterone (11-keto-Testo), 1-dehydrotestosterone (Δ1-Testo), 4-androstene-3,17-dione (Δ4-dione), 4-androsten-11β-ol-3,17-dione (11β-OH-Δ4-dione), 4-androsten16α-ol-3,17-dione (16α-OH-Δ4-dione), epitestosterone (epiTesto), androsterone, epiandrosterone, 5β-androstan-3α-ol-17-one (5β-A-3α-17-one), 5β-androstan-3β-ol-17-one (5β-A-3β-17one), 5α-androstan-3,17-dione (5α-A-dione) and 5β-androstan3,17-dione (5β-A-dione). Other chemicals and solvents (of analytical grade) were purchased from the following sources: ammonium acetate, sodium acetate, phosphate buffer (0.01 M) and sodium hydroxide from Sigma-Aldrich; acetonitrile and ethyl acetate from Scharlau Chemie S.A. (Barcelona, Spain), and ethanol and acetic acid from Merck (Briare-Le-Canal, France). Ultrapure water, obtained using a Milli-Q system (Millipore, Saint-Quentin-en-Yvelines, France), was used for ex vivo preparations and for the preparation of HPLC mobile phases. Dulbecco’s modified Eagle’s medium and L-glutamine were obtained from GibcoTM (Cergy-Pontoise, France). Antibiotics (streptomycin/penicillin and gentamycin) and fungizone, used in the culture media, were purchased from Sigma-Aldrich. Pig Ear Skin Short-Term Cultures Fresh Skin The preparation and incubation conditions of pig skin explants have been previously detailed by Jacques et al. [17]. Briefly, skin cultures were developed from ears of domestic pigs (Pietrain
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189
breed, 6-month-old females) as these were easily obtained from a local slaughterhouse. After cleaning and shaving, the skin was immediately excised with a scalpel and sectioned at a thickness of 500 μm and punched in 28-mm-diameter discs. Skin punches were placed dermal side down in 23-mm-diameter inserts in 6-well plates prefilled with 1.5 ml of culture medium per well at 37 ° C in a CO2 air incubator. [14C]-testosterone (8,333 Bq/sample), which was adjusted with unlabeled testosterone to reach the required doses of 50, 100, 200, 400, 600 and 800 nmol (corresponding to 3.03, 6.06, 12.12, 24.25, 36.39 and 48.52 μg/cm2, respectively), was diluted in 60 μl of phosphate buffer/ethanol (1:1, v/v) and applied to the skin surface. Culture media collected at 24 and 48 h and at the end of the experiment (72 h) were stored at –20 ° C until analysis. All incubations were carried out in triplicate.
Frozen Skin The influence of skin viability on percutaneous absorption was investigated by comparing testosterone diffusion in fresh explants and in identical skin preparations which had previously been frozen (2 months at –20 ° C). After this enzyme inactivation period, skin punches were incubated similarly as fresh skin with 50, 200 and 800 nmol [14C]-testosterone (8,333 Bq) applied to the skin punches, respectively.
Compartmental Analysis of Radiolabeled Testosterone Compartmental analysis was carried out as previously described [17–19]. Briefly, skin surfaces, tissue culture inserts and wells were washed twice with methanol. Testosterone and metabolites were extracted from the skin after homogenization with a Polytron homogenizer (Kinematica, Luzern, Switzerland) in methanol/phosphate buffer at pH 7.4 (2: 1; v/v) and centrifuged. Radioactivity in culture media, skin extracts and washing solutions was quantified using a scintillation counter (Tri-Carb; Perkin-Elmer, Waltham, Mass., USA). Microsomal Fractions Liver microsomes obtained from pools of 10 human male and female donors were purchased from TEBU (Le-Perray-en-Yvelines, France). Human skin microsomes (pool of 2 donors) were purchased from Biopredic (Rennes, France). Pig liver and skin microsomes were prepared from domestic pigs as previously described by Jacques et al. [17, 18] and Zalko et al. [19]. All microsomes were stored at –80 ° C until use. The protein content of microsomal preparations was determined according to the method of Lowry et al. [21].
Metabolism of Testosterone by Skin and Liver Microsomes Radiolabeled [14C]-testosterone (3,333 Bq) fortified with unlabeled testosterone to reach the required concentrations (1, 5, 10, 50, 100, 150 and 200 μM, respectively) was incubated for 20 min at 37 ° C under shaking with 2 mg of microsomal protein from human liver or pig liver, respectively. Similar conditions were used for experiments carried out using human and pig skin microsomes with two testosterone concentrations (1 and 5 μM). Incubations were performed in a final volume of 1 ml of 0.1 M sodium phosphate buffer, 5 mM MgCl2 (pH 7.4) containing an NADPH-generating system (1.3 mM NADP, 5 mM glucose-6-phosphate and 2 IU glucose-6-phosphate dehydrogenase). All pig microsome incubations were performed in triplicate. Blank samples without an NADPH
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generating system were used as controls for non-NADPH-mediated testosterone transformations. At the end of the incubation period, the reaction was stopped by protein precipitation with the addition of 3 vol methanol and centrifugation for 10 min at 8,000 g. Supernatant aliquots (500 μl) were concentrated under a gentle nitrogen stream and analyzed by HPLC coupled to online radioactivity detection. Metabolite identification was performed by MS analysis. Analysis of Testosterone by Radio-HPLC For all samples (culture media, skin extracts and microsomal incubations), 3 vol methanol were added. Samples were stirred for 1 min and then centrifuged. Supernatants were concentrated under a gentle nitrogen stream and metabolites were separated by radio-HPLC. The HPLC system consisted of a Spectra P1000 pump (Thermo Separation Products, Les Ulis, France) associated with a 250 × 4 mm (5-μm) Kromasil-C18 column (Bischoff, Montluçon, France) protected by a Kromasil-C18 guard column (Interchim, Montluçon, France). Mobile phases used at a flow rate of 1 ml/min consisted of ammonium acetate (20 mM, pH 3.5) and acetonitrile (A). The temperature of the column was maintained at 35 ° C and the following gradients were used: 0 min, 20% A; 35 min, 40% A; 50 min, 60% A; 55 min, 100% A; 60 min, 20% A. [14C]-testosterone and related metabolites were monitored by online radioactivity detection using a Flo-one A500 (Radiomatic, LaQueue-Lez-Yvelines, France) and Flow-scint II (Packard Instruments, Downers Grove, Ill., USA) as the scintillation cocktail. Testosterone and its metabolites were quantified by integrating the area of the radiochromatographic peaks. The limit of detection is 20 dpm and corresponds to three times the noise signal on the radiochromatogram and the limit of quantitation is 70 dpm and corresponds to ten times the noise signal on the radiochromatogram.
Structural Characterization of Testosterone Metabolites Metabolites were isolated using HPLC separation coupled to metabolite collection using a Gilson model 201/202 fraction collector (Gilson France, Villiers-Le-Bel, France). Radioactivity in the different fractions was determined by direct counting of sample aliquots on the Packard scintillation counter. Fractions of interest were pooled and dried under a gentle nitrogen stream for MS analyses. For the less water-soluble metabolites (typically those with a retention time above 30 min in HPLC), no derivatization step was required prior to gas chromatography (GC) MS analysis. Such samples, once isolated, were directly dissolved in 40 μl of methanol before injection. For the other metabolites, including OH-Testo and OH-Δ4-dione, a derivatization step was required prior to GCMS, to ensure an efficient detection. For this purpose, 50 μl of N,Obis(trimethylsilyl)trifluoroacetamide supplemented with 1% trimethylchlorosilane (Grace Davison Discovery Sciences, Deerfield, Ill., USA) were added to the samples. The derivatization reaction proceeded for 12 h at 60 ° C. Then, reaction mixtures were evaporated to dryness under a nitrogen stream and immediately dissolved with 40 μl of hexane. GC-MS analyses were performed on a Trace GC 2000 device associated with a Polaris ion trap mass analyzer (Thermo Scientific, Les Ulis, France). The injection volume was 2 μl in the splitless mode at 270 ° C. Chromatographic separation was achieved on an Optima 5 Accent capillary column (30 m × 0.25 μm, 0.25 μm) from Macherey-Nagel (Hoerdt, France). Helium was used as the carrier
Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
Diffusion of testosterone and its metabolites (nmol/cm2)
Fig. 1. Diffusion of testosterone in culture
90 72 h 48 h 24 h
80 70 60 50 40 30 20 10 0
50
100 200 400 600 Dose of testosterone applied on skin explants (nmol)
media as a function of the incubation time for different doses (means ± SD, n = 3).
800
Table 1. Radioactivity distribution in media, skin and washing solutions 72 h after the beginning of the experi-
ment for each testosterone dose applied on the skin surface Testosterone dose 50 nmol
100 nmol
200 nmol
400 nmol
600 nmol
800 nmol
Skin surface Skin Culture media Culture insert Well washing
0.7±0.4 9.9±0.8 84.2±15.7 0.6±0.1 3.0±0.2
0.6±0.1 16.1±5.2 86.5±4.6 0.9±0.2 3.6±0.8
0.7±0.1 19.0±6.8 80.5±4.0 0.9±0.1 3.8±0.8
1.0±0.2 25.1±5.8 72.5±3.5 1.1±0.3 3.4±1.1
1.5±0.4 32.0±1.7 59.0±6.2 1.1±0.2 3.3±1.1
1.9±2.3 32.7±1.9 41.3±9.6 1.7±1.6 1.6±0.4
Total
98.5±16.6
107.4±10.6
104.9±12.1
103.3±9.0
97.4±8.2
79.3±12.7
Results are expressed as percent of the applied radioactivity (means ± SD, n = 3).
gas at a flow rate of 1 ml/min. The following oven program was used: 40 ° C for 1 min, then 40–250 ° at 25 ° C/min (8.4 min), 250– 280 ° at 5 ° C/min (6 min) and a final hold time of 10 min at 280 ° C. The injector and GC-MS interface were set at 250 ° C. Positive electron ionization (EI) mass spectra (70 eV) were generated at a source temperature of 220 ° C. Spectra were acquired from m/z 60–550.
Results
Distribution of Testosterone in Pig Ear Skin: Short-Term Cultures Table 1 summarizes the compartmental distribution of radioactivity (expressed as percent testosterone equivalent) at 72 h. Mass balance recovery for 50–600 nmol testosterone ranged between 97.4 and 107.4%. The mass balance met the requirements of the Scientific Committee Cutaneous Testosterone Metabolism
on Consumer Safety guidelines. Only at the highest dose (800 nmol), mass balance recovery dropped slightly below 85%. The radioactivity remaining on the surface of the skin at the end of the experiments (72 h) ranged between 0.6 and 1.9% of the applied radioactivity. In the skin itself, radioactivity levels gradually increased from 9.9 (50 nmol testosterone) to 32.7% (800 nmol testosterone). Conversely, the main part of the radioactivity was recovered in culture media, but with decreasing percentages for testosterone doses of 50 (84.2%) to 800 nmol (41.3%). Residual radioactivity recovered from inserts and wells never exceed (together) 4.7% of the applied doses. Figure 1 details the diffusion of testosterone in culture media as a function of the incubation time (24, 48 and 72 h) and for the different doses tested. Whatever the apSkin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
191
100
Testosterone Metabolites
90
Radioactivity (%)
80 70 60 50 40 30 20 10 0
50
a
200
800
50
Surface skin
200
800
50
Skin
200
800
Medium
90 Testosterone Metabolites
80
Radioactivity (%)
70 60 50 40 30 20 10
Fig. 2. Comparison of radioactivity distri-
bution (testosterone + metabolites, 72 h) in fresh (a) and frozen skin explants (b). Results are expressed as percent of the applied radioactivity (mean ± SD, n = 3).
0
b
50
200 Surface skin
plied dose, the flux of testosterone (sum of the parent compound and metabolites, based on culture medium counts) decreased during each successive 24-hour period. For instance, when applying 600 nmol of testosterone, 40.1, 24.5 and 20.5 nmol of testosterone equivalent/cm2 were recovered in the culture medium following the three 24-hour time periods, respectively. At this concentration, testosterone flux was 1.67, 1.02 and 0.99 nmol/h/cm2, respectively. This decrease in testosterone flux was likely due to the progressive depletion of available testosterone, and this trend was confirmed for all the other concentrations tested. Fresh versus Frozen Skin Figure 2 presents the compartmental analysis of 3 different doses of testosterone and its metabolites for fresh (fig. 2a) and skin frozen for 2 months prior to the experi192
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
800
50
200 Skin
800
50
200
800
Medium
ments (fig. 2b). For frozen skin, the mass balance was found to meet OECD guidelines, but the quantity of radioactivity remaining at the surface of the skin at 72 h was slightly higher than in experiments carried out with fresh skin. In the skin itself, the percentage of radioactivity was also higher for frozen skin. Conversely, the diffusion of testosterone within fresh skin, estimated by the percentage of radioactivity in culture media, was higher than in frozen skin whatever the dose of testosterone applied. For instance, in 50-nmol assays, 84.2 ± 5.1 and 75.4 ± 3.8% of the applied dose was recovered in culture media of fresh and frozen skin, respectively. Culture media as well as skin extracts were analyzed by radio-HPLC (fig. 3). When frozen skin was used, the radioactivity recovered in culture media and skin corresponded to the parent testosterone only, indicating Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
2,750
Testosterone
2,500 2,250
DPM
2,000 1,750 1,500 1,250 1,000 750 500 250 0
0
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30
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40
50
60
Time (min) 1,000
VIII
900 800 700
XIII
DPM
600 XI
500
XII
400
XIII’
300 Testosterone
200 100
Fig. 3. Typical radiochromatographic pro-
files obtained from culture media using frozen (a) and fresh skin explants (b) exposed to [14C]-testosterone (200 nmol, 72 h).
0
b
I
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5
II
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IV V’
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65
that no biotransformation had taken place. Conversely, when using fresh skin explants, a high proportion of the radioactivity, both in culture media and skin extracts, corresponded to testosterone metabolites, which eluted either before or after testosterone under our HPLC conditions. A representative radiochromatogram displayed in figure 3 corresponds to the analyses of 72hour samples of culture media, frozen (fig. 3a) and fresh (fig. 3b) skin explants, respectively, for incubations carried out with 200 nmol of testosterone. Fourteen peaks were detected in the culture media. All were also detected in skin extracts. At the end of the experiment,
metabolites in culture media ranged from 81.4 [50-nmol assays] to 39.9% [800-nmol assays] of the dose, while the proportion of metabolites in skin extracts gradually increased from 9.9 [50-nmol assays] to 32.6% [800-nmol assays]. For the 50-nmol dose, peak VIII was the major metabolite detected in culture medium samples (75.9 ± 4.7%) as well as skin extracts (88.1 ± 1.6%). Other metabolites detected in culture media and in skin extracts accounted, all together, for 22.4 ± 4.9 and 13.1 ± 3.1% of the radioactive dose, respectively. Only low rates of unmetabolized testosterone were recovered in culture me-
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Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
193
Table 2. GC-MS (EI) identification of testosterone metabolites formed in incubations of testosterone carried out with liver microsomes or pig ear skin explants (for which both culture media and skin extracts were analyzed)
Peak No.
Metabolite
Radio-HPLC min
GC RT min
Standard, GC RT min
M+· ions m/z
I II III IV Vb V’c VI VII VIII IXb IX’c X Testo XI XII XIII’b XIII
7α-OH-Testo 6β-OH-Testo 16α-OH-Testo OH-Δ4-dione OH-Testo OH-Δ4-dione 16α-OH-Δ4-dione OH-Testo OH-Δ4-dione OH-Testo OH-Δ4-dione Δ6-Testo testosterone androstenedione epiandrosterone 5α-A-dione androsterone
9.0 12.0 14.0 16.0 18.0 17.5 20.0 21.0 25.5 28.0 27.9 33.0 35.4 40.0 43.0 47.0 48.0
14.20 14.23 15.79 14.92 15.70 15.70 15.16 14.49 15.67 15.81 15.79 14.61 14.30 14.14 13.30 13.59 13.24
7α-OH-Testo-TMS2 (14.20) 6β-OH-Testo-TMS2 (14.23) 16α-OH-Testo-TMS2 (15.79) standard not available standard not available standard not available 16α-OH-Δ4-dione-TMS (15.16) standard not available standard not available standard not available standard not available standard not availabled testosterone (14.30) androstenedione (14.14) epiandrosterone (13.30) 5α-A-dione (13.60) androsterone (13.24)
448.0a 448.0a 448.0a 374.0a 448.0a 374.0a 374.0a 448.0a 374.0a 448.0a 374.0a 286.1 288.2 286.1 290.2 288.1 290.1
a Derivatized
with N,O-bis(trimethylsilyl)trifluoroacetamide-trimethylchlorosilane (1%). observed in pig ear skin explants. c Only observed in microsomal incubations. d Identification made on the basis of Brown and Djerassi [22] in 1981. RT = Retention time. b Only
dia as well as skin extracts, irrespective of the dose applied. These results demonstrated extensive biotransformation of the model substrate. Structural Characterization of Skin Testosterone Metabolism Metabolism of Testosterone by Short-Term Skin Cultures: Qualitative Analysis Metabolites collected from culture media and skin extracts were analyzed by GC-MS using EI ionization. GCMS data are summarized in table 2. The most polar metabolites, which correspond to compounds eluted before 30 min, did require a derivatization step prior to GC-MS analysis. Trimethylsilyl derivates (TMS) of hydroxylated metabolites produced characteristic losses of a methyl radical and HOSi(CH3)3 from M+ · ions in EI MS. Accordingly, peaks I, II, III, V, VII and IX were all identified as testosterone hydroxylated metabolites (OH-Testo), since their respective mass spectra displayed two derivatized hydroxyl functions, as well as fragment ions in full concordance with such structure (data not shown). Analyses performed with standard testosterone metabolites by GC-MS further allowed to detail the structural character
194
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ization of these metabolites. Thus, peaks I, II and III were attributed to 7α-OH-Testo, 6β-OH-Testo and 16α-OHTesto, respectively. The position of the hydroxyl group of other OH-Testo metabolites could not be determined on the basis of the comparison with available standards. Metabolites corresponding to peaks IV, VI and VIII were identified as hydroxyandrostenediones (OH-Δ4-diones), since their respective mass spectra showed one derivatized function and a fragmentation pattern consistent with a OH-Δ4-dione structure. Peak VI was identified as the 16α-Δ4-dione following comparison with the corresponding authentic standard. The hydroxylation position of other OH-Δ4-dione metabolites could not be determined on the basis of standard analyses. Alternatively, metabolites eluted after 30 min were submitted directly to GC-MS analyses (i.e. without TMS derivatization). The compound which eluted at 35.4 min in radio-HPLC was confirmed to be testosterone. The mass spectrum of peak X displayed M+ · ions at m/z 286 with a characteristic fragmentation pattern of 17β-hydroxyandrosta-4,6-dien-3-one (Δ6-Testo) and diagnostic fragment ions detected at m/z 136 [22]. The mass spectrum and GC retention time of peak XI were both
Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
Metabolites formed (nmol/h)
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2.0 1.5 1.0 0.5 0 0
100
200
300
400
500
600
700
800
Testosterone dose applied on skin (nmol)
Fig. 4. Testosterone metabolite production at 72 h as a function of the testosterone dose applied on skin explants. Calculations are based on concentrations measured in culture media and skin extracts (means ± SD, n = 3).
consistent with the results obtained for the androstenedione standard. Similarly, GC-MS analyses of peaks XII, XIII and XIV were compared with the analyses of the corresponding standards, allowing the attribution of these metabolites to epiandrosterone, 5α-A-dione and androsterone, respectively, as shown in table 2. Metabolism of Testosterone by Skin Short-Term Cultures: Quantitative Analysis Production rates (in nmol/h) of testosterone metabolites in skin short-term cultures were calculated based on radio-HPLC analyses. Kinetic curves were plotted as a function of the testosterone dose (fig. 4). Peak VIII (OH-Δ4-dione) was the major metabolite produced by skin short-term cultures. For this metabolite, a saturation occurring at around 400 nmol of testosterone was observed and followed Michaelis-Menten kinetics, with Vm and Km values of 3.09 ± 0.33 nmol/h/mg and 165.4 ± 55.4 μM, respectively. The formation rate of all other metabolites (epiandrosterone, androsterone, androstenedione, 5α-A-dione, OH-Testo, 6β-OH-Testo, 7α-OH-Testo, 16α-OH-Testo, 16α-Δ4-dione and OHΔ4-diones) also followed Michaelis-Menten kinetics, and, as observed for OH-Δ4-dione, seemed to reach saturation around a testosterone dose of 400–800 nmol. However, for these metabolites, the range of concentrations selected for the study did not allow a precise determination of the saturating dose and thus the calculation of kinetic constants. Cutaneous Testosterone Metabolism
Testosterone Metabolism by Liver and Skin Microsomes of Pig and Human Origin Metabolism of Testosterone by Liver and Skin Microsomes: Qualitative Analysis In incubations of [14C]-testosterone with microsomes (either from pig liver, pig skin, human liver or human skin), always >90% of the radioactivity put in incubations was recovered at the end of the experiments. In our analytical conditions, the retention time of testosterone was 35.4 min. Qualitatively, similar metabolic profiles were observed for incubations carried out with human or pig microsomal fractions. Using liver microsomes, a large proportion of the radioactivity corresponded to testosterone metabolites >54.5%. The radio-HPLC profiles obtained for these incubations were also very similar to the radio-HPLC profile obtained for fresh pig ear skin explants. For skin microsome incubations, only three groups of metabolites were detected (peaks III, XI and XII). Finally, none of the metabolites was observed when no NADPH-generating system was added to the incubations (data not shown), demonstrating that their formation was cytochrome P450 dependent. Structural Characterization of Testosterone Metabolites Some metabolites displayed identical HPLC retention times as those produced by skin culture (peaks I–IV, VI– VIII, X–XII). All GC-MS results are summarized in table 2. Metabolites eluting before 30 min in our radioSkin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
195
HPLC system (peaks I–IX’) required to be derivatizated prior to their characterization by GC-MS. Metabolites corresponding to peaks I, II, III and VII were identified as OH-Testo, similarly to the corresponding metabolites produced by pig skin. Comparison with GC-MS analyses of standard metabolites allowed to identify peaks I, II and III as 7α-OH-Testo, 6β-OH-Testo and 16α-OH-Testo, respectively. The hydroxylation position of other OHtestosterone metabolites was not determined on the basis of available standards. OH-Testo metabolites V and IX produced by skin short-term culture were not detected in microsomal incubations. Conversely, OH-Δ4-dione metabolites were characterized (peaks V’ and IX’). Peak VI was confirmed to be 16α-Δ4-dione. We were unable to determine the position of the hydroxyl group of other OH-Δ4-dione metabolites. Metabolites eluted with a retention time >30 min were directly analyzed by GC-MS. Retention times and mass spectra allowed to attribute peaks X and XI to Δ6-Testo and androstenedione, respectively. Similarly, GC-MS analyses of peaks XII and XIV compared with analyses of appropriate standards permitted to identify these metabolites as epiandrosterone and androsterone, respectively (table 2). Finally, metabolite XIII was only observed in pig ear skin samples and was identified as 5α-A-dione by GCMS analysis. Radio-HPLC and GC-MS analyses of incubations with liver microsomes showed that the same metabolites were generated in pig and human samples (table 2). Due to the low amount of testosterone metabolites formed by pig and human skin microsomes, their identification was based only on HPLC retention times comparison with authentic standards and/or previously identified biotransformation products. For both species, the three groups of metabolites detected by radio-HPLC had retention times which were similar to the three of the metabolites produced by liver microsomes (table 2), respectively, namely 16α-OH-Testo (peak III), androstenedione (peak XI) and epiandrosterone (peak XII). Metabolism of Testosterone by Liver and Skin Microsomes: Quantitative Analysis Testosterone metabolic rates were investigated over a substrate concentration range from 1 to 200 μM for pig liver microsomes. Human liver and skin microsomes were incubated with two testosterone concentrations (1 and 5 μM). The main metabolites formed by liver and skin microsomes from humans and pigs are represented in the figure 5. For humans as well as pigs, overall testosterone biotransformation rate was lower in the skin com196
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
pared to the liver. For example, at a substrate concentration of 5 μM, 2.73 ± 0.88 nmol of testosterone metabolites were formed in pig liver microsomal incubations, whereas only 0.26 ± 0.08 nmol were produced with pig skin microsomes. The metabolic rate of testosterone by skin microsomes was approximately 10 times lower than that with liver microsomes. Major metabolites detected in liver microsome incubations were also detected in skin microsome incubations (16α-OH-Testo, androstenedione and epiandrosterone). Other metabolites, which were identified in liver microsome incubations, could not be detected in skin microsome incubations, probably due to analytical limits of detection (20 dpm for radio-HPLC).
Discussion
In this work, the absorption and metabolic capabilities of a pig ear skin system were studied to investigate the specific hydroxylation activities expressed in this ex vivo skin model and to examine the impact of testosterone biotransformation on its bioavailability. Testosterone is an endogenous hormone with a complex metabolism and a model compound recommended by the OECD. The low molecular weight (288) and relatively lipophilic nature (log Kow = 3.3) of testosterone are factors that improve skin penetration [23]. Furthermore, the pattern of testosterone hydroxylation is commonly used to characterize phase I metabolism by different isoforms of cytochrome P450 in the rat liver. Several models enabling the study of fate of testosterone are currently available, but there are little data taking biotransformation issues for this molecule into account. The penetration (skin barrier function), absorption and biotransformation capabilities (phase I and II metabolic activities) of the ex vivo pig skin model were investigated using labeled testosterone on fresh (viable) and frozen (metabolically inactivated) skin. Compared to incubations carried out with fresh skin, testosterone absorption seemed to be weaker with frozen skin, but the observed difference was not statistically significant. Conversely, a tendency was observed for the amount of radioactivity remaining at the surface of the skin by the end of the experiments, with higher values recorded for the surface of the skin and in the skin, itself. However, from a qualitative point of view, striking differences were observed between fresh and frozen skin incubations, with only parent testosterone being recovered in culture media for the latter system. Using fresh skin explants, the major part of the radioactivity recovered in culture media as well Jacques/Perdu/Jamin/Cravedi/Mavon/ Duplan/Zalko
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duced by liver and skin microsomes from pigs (a) and humans (b; means ± SD, n = 3).
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as in the skin itself corresponded to metabolites (>95%). The formation of metabolites increased with the concentration of testosterone, and a beginning of saturation was observed only after application of 400 nmol of testosterone or more. Metabolism was shown to follow MichaelisMenten kinetics. These data demonstrate that testosterone was extensively metabolized by the skin, and that the barrier effect of the ex vivo pig ear skin model was preserved over the whole duration of the experiment. This is fully consistent with our previous work on benzo(a)pyrene, with the same ex vivo pig ear skin model [17, 24]. When using viable epidermis, the rate of transport of the parent molecule is often the limiting factor. In the case of
a lipophilic compound, and if the parent molecule is metabolized into a more hydrophilic compound, percutaneous absorption of the metabolite can be greatly enhanced and actually can be faster than the absorption of the parent compound [25]. In our study, it is remarkable that >95% of the testosterone diffused into the skin are in the form of metabolites. Testosterone was biotransformed into androstenedione, 5α-A-dione, androsterone, epiandrosterone, Δ6Testo, OH-Δ4-diones and OH-Testo, which oxidated at different positions of the molecule (fig. 6). Combined with the kinetic parameters detailed in the results section, this demonstrates that oxidative enzymes were function-
Cutaneous Testosterone Metabolism
Skin Pharmacol Physiol 2014;27:188–200 DOI: 10.1159/000351683
197
OH H H O
H OH
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5α-Dihydrotestosterone (290)
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