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Original Paper

Skin Pharmacol Physiol 2015;28:124136DOI: 10.1159/000365730

The Influence of the Acyl Chain on theTransdermal Penetration-EnhancingEffect of Synthetic Phytoceramides

Lieselotte Veryser a Jente Boonen a Lien Taevernier a Joren Guillaume b Martijn Risseeuw b Syed Nisar Hussain Shah d Nathalie Roche c Serge Van Calenbergh b Bart De Spiegeleer a a Drug Quality and Registration Group, and b Laboratory of Medicinal Chemistry, Faculty of PharmaceuticalSciences, Ghent University, and c Department of Plastic and Reconstructive Surgery, University Hospital Ghent,Ghent , Belgium; d Faculty of Pharmacy, Bahauddin Zakariya University, Multan , Pakistan

PCER10 (4.84 0.79), but none of them had an influence onibuprofen. Conclusion: The investigated PCERs exhibited apenetration-enhancing effect on caffeine and testosteronebut not on ibuprofen. 2014 S. Karger AG, Basel

Introduction

The skin has an important protective function againstenvironmental damage, in which the outermost layer, thestratum corneum (SC), plays an important role. The 10-to 20-m-thick layer acts as a penetration barrier, whichprotects the body against bacterial, enzymatic or chemical

impacts and prevents excessive water loss. The SC ismainly composed of corneocytes surrounded by multila-mellar organized lipids [1, 2] . Ceramides are the majorlipid class of the extracellular matrix of the SC and are es-sential components of the SC barrier function. They arefound in lamellar bodies of differentiating keratinocytes[3]. Ceramides not only contribute to the skin barrierfunction but also play physiological roles in signal trans-duction and cell regulation. When the cell membrane is

Key WordsPhytoceramide Azone Penetration enhancer Skinpenetration

AbstractBackground/Aims: The skin has become very attractive as aroute for drug administration. Optimization of topical drugformulations by the addition of penetration enhancers mayfacilitate the passage of drugs through the stratum corne-um. Methods: In this paper, the skin penetration effect ofphytosphingosine and 9 derived phytoceramides (PCERs) on3 transdermal model drugs (i.e. caffeine, testosterone, ibu-profen) was investigated via Franz diffusion cell experiments

using split-thickness human skin. Azone was included as apositive control. Results: The main finding in our study wasthat the PCERs exerted a compound-dependent penetra-tion-enhancing effect. Some of the investigated PCERs ex-hibited a penetration-enhancing ratio of more than 2 (mean SE): for caffeine PCER1 (2.48 0.44), PCER2 (2.75 0.74),PCER3 (2.62 0.93) and PCER6 (2.70 0.45) and for testos-terone PCER1 (2.08 0.56), PCER2 (2.56 0.13), PCER3 (3.48),PCER4 (2.53), PCER5 (2.04 0.14), PCER6 (2.05 0.48) and

Received: December 12, 2013Accepted: July 2, 2014Published online: December 16, 2014

Bart De SpiegeleerDrug Quality and Registration (DruQuaR) GroupFaculty of Pharmaceutical Sciences, Ghent UniversityOttergemsesteenweg 460, BE9000 Ghent (Belgium)E-Mail Bart.DeSpiegeleer @ UGent.be

2014 S. Karger AG, Basel16605527/14/02830124$39.50/0

www.karger.com/spp


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Acyl Chain and Transdermal

Penetration-Enhancing Effect of PCERs

Skin Pharmacol Physiol 2015;28:124136

DOI: 10.1159/000365730

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altered during several biological processes, ceramide lev-els increase, and they induce cell differentiation and/orapoptosis and reduce cell proliferation [4, 5] .

Chemically, a ceramide (2-N-acylsphingosine) is anacyclic 2-amino-1,3-diol, composed of a sphingoid basewith a double bond between carbons 4 and 5, which is

linked to a fatty acid through an amide bond. Depend-ing on the nature of the sphingosine moiety and the fattyacid chain, 12 different classes of ceramides can be dis-tinguished in human skin [6, 7] . Ceramides with a sat-urated sphingosine base containing an additional hy-droxyl group at carbon 4 are known as phytoceramides(PCERs) [3].

Recently, ceramides have gained much attention aspossible transdermal penetration enhancers, which arecapable of increasing the amount of drugs permeatingthrough the skin by reducing the barrier resistance of theSC. Skin penetration enhancers are present in several der-matological and cosmetic products to enhance the localand/or systemic absorption of the active compound. Onlya few fragmental studies demonstrated the penetration-enhancing properties of selected ceramides [812] . Inthese studies, the effect of the ceramide acyl chain lengthon the skin permeability was investigated [13] , indicatingthat short-chain ceramides increase the skin permeabilityof theophylline and indomethacin and exhibit a maximaleffect with an acyl chain length between 4 and 6 carbons.However, studies using PCERs are lacking. This led us toassess the penetration-enhancing effect of phytosphingo-sine and a series of 9 PCERs with different acyl chains, viatransdermal Franz diffusion cell (FDC) experiments onthe 3 transdermal model compounds obtained by classi-fication and regression trees, i.e. caffeine, testosteroneand ibuprofen [14] . The well-known penetration enhanc-er azone served as a positive control. In our previous stud-ies, these model compounds were already investigated inthe presence or absence of other penetration enhancers,such as spilanthol [15, 16] .

Materials

Products ExaminedCaffeine and ibuprofen (Ph. Eur. grade) were obtained from

ABC Chemicals (Vemedia, Wouters-Brakel, Belgium), while testos-terone ( 99% grade) was purchased from Sigma-Aldrich (Bornem,Belgium). The investigated PCER types 29 (PCER2PCER9) andazone were prepared in-house (see section Synthesis of Phytosphin-gosine and PCERs). Phytosphingosine (PCER1) and salicyloyl phy-tosphingosine (PCER10) were obtained from Evonic Industries (Es-sen, Germany). The structures of the PCERs are presented in table 1,and they will further be indicated as PCER1PCER10.

Table 1. Structures of the investigated phytosphingosine (PCER1),PCERs and azone (AZ)

Code Structure

PCER1 NH 2C 14 H 29

OH

OH

HO

PCER2NH O H

CH 3O

C 14 H 29OH

HO

PCER3NH O H

CH 3O

C 14 H 29OH

HO

PCER4NH O H

CH 3O

C 14 H 29HO

PCER5N H O H

CH 3O

C 14 H 29OH

HO

PCER6NH OH

CH 3O

C 14 H 29OH

HO

PCER7NH OH

CH 3O

C14

H29

OH

HO

PCER8NH OH

CH 3O

C 14 H 29

OH

HO

PCER9

NH OH

O

C 14 H 29

OH

HO

PCER10

NH OH

O

C 14 H 29

OH

HO

HO

AZ

N

O

CH 3


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Chemicals and ReagentsUltrapure water (H2 O) of 18.2 M cm quality was produced

by an Arium 611 purification system (Sartorius, Gttingen, Ger-many), while triethylamine, methanol, sodium carbonate, 1-bro-mododecane, octanoic acid, tetradecanoic acid, benzoylchlorideand -caprolactam came from Acros Organics (Geel, Belgium). Ab-solute ethanol (99.8% V/V), acetic anhydride, toluene, pyridine,dichloromethane (CH2 Cl2 ), thionyl chloride, butyric acid, tetrahy-drofuran, hexanoic acid, sodium hydride, decanoic acid, dodeca-

noic acid, diethyl ether, sodium hydrogen carbonate, sodium sul-phate, sodium hydroxide and ethyl acetate were purchased fromSigma-Aldrich. 0.01M phosphate-buffered saline (PBS) was alsopurchased from Sigma-Aldrich and prepared according to the in-structions of the supplier. High-performance liquid chromatogra-phy (HPLC) gradient grade methanol (MeOH), acetonitrile andmagnesium sulphate came from Fisher Scientific (Erembodegem,Belgium), while formic acid (FA) was bought from Riedel-de Han(Seelze-Hannover, Germany). Denaturated ethanol (up to 5%ether) came from Chem Lab (Zedelgem, Belgium) and bovine se-rum albumin (BSA) was supplied by Merck (Darmstadt, Germany).

Methods

Synthesis of Phytosphingosine and PCERsD -ribo -Phytosphingosine (PCER1) was transformed into PCER2

by treatment with a 1: 1 mixture of acetic anhydride and pyridine,followed by hydrolysis of the acetate esters. PCER3PCER9 wereprepared under Schotten-Baumann reaction conditions using theappropriate acyl chlorides. The latter were obtained by treating thecorresponding fatty acids with thionyl chloride (fig. 1).

Precoated Macherey-Nagel SILG/UV254 plates were used forthin-layer chromatography, and spots were examined under UVlight at 254 nm and further visualized by sulphuric acid-anisalde-hyde spray. Preparative column chromatography was performed onBiosolve silica gel (4063 m, 60 ). Nuclear magnetic resonance(NMR) spectra were obtained with a Varian Mercury 300 Spectrom-eter (Palo Alto, Calif., USA). Chemical shifts are given in parts permillion () relative to the residual solvent signals, in the case of deu-terated chloroform: = 7.26 p.p.m. for1 H, in the case of pyridine-d5:= 8.74, 7.58 and 7.22 p.p.m. for1 H. Exact mass measurements wereperformed on a Waters (Milford, Mass., USA) LCT Premier XETOF equipped with an electrospray ionization interface (ESI). Sam-ples were infused in an acetonitrile/FA (1,000: 1) mixture at 10 ml/min. All reactions were performed using starting materials obtainedfrom commercial sources without further purification. Phytosphin-gosine and all obtained PCERs had a purity of >95% (NMR).

N-[(2S ,3S ,4R )-1,3,4-Trihydroxyoctadecan-2-yl]acetamide(PCER2)A solution of phytosphingosine (1 g, 3.15 mmol PCER1) in 40

ml acetic anhydride/pyridine (1: 1) was stirred for 24 h at roomtemperature. The reaction was quenched under ice-cold condi-tions with saturated sodium hydrogen carbonate solution and ex-tracted with CH 2 Cl2 . The organic layer was washed with saturatedsodium hydrogen carbonate and brine, dried over sodium sul-phate, filtered and evaporated to dryness to afford the crude phy-

tosphingosine tetra-acetate. This crude product was dissolvedin MeOH (20 ml) and triethylamine (10 ml) and was stirred for4 days at room temperature. The solvents were removed under re-duced pressure, and the residue was purified by column chroma-tography (CH 2 Cl2 :MeOH 9: 1) to give PCER2 (0.63 g, 56% yield)as a white solid.

1 H-NMR (300 MHz, pyridine): = 0.88 (t, J= 6.5 Hz, 3 H, CH3 ),1.151.51 (m, 22 H, CH2 ), 1.571.80 (m, 1 H, H-6a), 1.812.07 (m2 H, H-5a, H-6b), 2.13 (s, 3 H, CH3 ), 2.173.25 (m, 1 H, H-5b),4.234.31 (m, 1 H, H-4), 4.334.39 (m, 1 H, H-3), 4.48 (d, J= 5.1Hz, 2 H, CH2-1), 5.10 (dddd, J= 9.3 Hz and 5.0 Hz, 1 H, H-2), 6.19(br. s, 1 H, OH), 6.50 (br. s, 2 H, 2 OH), 8.60 (d, J= 8.5 Hz, 1 H,NH).

Exact mass (ESI-MS) for C20 H41 NO4 [M + Na] + found,

382.2931; calcd, 382.2928. General Schotten-Baumann Procedure: PCER3PCER9A 1-molar solution of fatty acid (2 equivalents) in thionyl

chloride was stirred overnight at room temperature. The mixturewas concentrated and co-evaporated with toluene to afford thecrude acid chloride. This was added dropwise to a 0.06-molarsolution of phytosphingosine (1 equivalent) in tetrahydrofu-ran/10% aqueous sodium carbonate solution (1: 1) at 0 C. Thereaction mixture was allowed to warm to room temperature andstirred overnight. After completion of the reaction, the aqueouslayer was extracted with CH 2 Cl2 , and the combined organic lay-ers were washed with 1 M sodium hydroxide and brine, dried overmagnesium sulphate, filtered and evaporated to dryness. The res-

idue was purified by column chromatography. PCER3PCER9were obtained using this general Schotten-Baumann procedurewith phytosphingosine (1 g, 3.15 mmol PCER1) and butyric acid,hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid,tetradecanoic acid and benzoylchloride, respectively. Columnchromatography (CH 2 Cl2 :MeOH 95: 5) gave PCER3 (671 mg,55%), PCER4 (555 mg, 42%), PCER5 (1.06 g, 76%), PCER6(181 mg, 12%), PCER7 (759 mg, 48%) and PCER9 (305 mg, 23%)as a white solid, while PCER8 (1.05 g, 63%) was a white electro-static powder.

HO

OH

OH

1

2 R = CH 33 R = (CH 2)2CH34 R = (CH 2)4CH35 R = (CH 2)6CH36 R = (CH 2)8CH37 R = (CH 2)10 CH38 R = (CH 2)12 CH39 R = Ph

a or bC14 H29

NH 2HO

OH

RO

OHC14 H29

NH

Fig. 1. Synthesis of PCER2PCER9. Re-agents and conditions: (a) first Ac 2 O/pyri-dine (1: 1), for 24 h; then triethylamine inMeOH, for 4 days, for No. 2; (b) firstRCOOH, SOCl2 , for 14 h; then No. 1, tetra-hydrofuran/10% aq. sodium carbonate(1: 1) 0 C room temperature, for 14 h, for

No. 39. Ph = Phenyl.


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N-[(2S ,3S ,4R )-1,3,4-Trihydroxyoctadecan-2-yl]butyramide(PCER3)1 H-NMR (300 MHz, CD 3 OD): = 0.92 (t, J = 6.7 Hz, 3 H, CH3 ),

0.98 (t, J = 7.4 Hz, 3 H, CH3 ), 1.291.41 (m, 24 H, CH 2 ), 1.501.74(m, 4 H, CH 2 ), 2.23 (t, J = 7.4 Hz, 2 H, CH 2-6), 3.503.61 (m, 2 H,H-4, H-1a), 3.683.82 (m, 2 H, H-3, H-1b), 4.12 (dd, J = 5.4 and10.0 Hz, 1 H, H-2).

Exact mass (ESI-MS) for C22 H45 NO4 [M + H] + found, 388.3427;calcd, 388.3421.

N-[(2S ,3S ,4R )-1,3,4-Trihydroxyoctadecan-2-yl]hexanamide(PCER4)1 H-NMR (300 MHz, pyridine): = 0.80 (t, J = 7.0 Hz, 3 H, CH 3 ),

0.830.93 (m, 3 H, CH 3 ), 1.171.55 (m, 26 H, CH 2 ), 1.641.86 (m,3 H, CH2 ), 1.872.03 (m, 2 H, CH 2 ), 2.192.32 (m, 1 H, H-5a), 2.43(t, J = 7.5 Hz, 2 H, CH2-2 ), 4.244.33 (m, 1 H, H-4), 4.364.43 (m,1 H, H-1a), 4.454.54 (m, 2 H, H-3, H-1b), 5.10 (dddd, J = 5.0 and9.6 Hz, 1 H, H-2), 6.17 (br. s, 1 H, OH), 6.53 (br. s, 2 H, 2 OH),8.45 (d, J = 8.4 Hz, 1 H, NH).

Exact mass (ESI-MS) for C24 H49 NO4 [M + Na] + found,438.3559; calcd, 438.3554.

N-[(2S ,3S ,4R )-1,3,4-Trihydroxyoctadecan-2-yl]octanamide(PCER5)1 H-NMR (300 MHz, pyridine): = 0.82 (t, J = 6.7 Hz, 3 H, CH 3 ),

0.850.95 (m, 3 H, CH 3 ), 1.091.53 (m, 30 H, CH 2 ), 1.652.03 (m,5 H, CH2 , H-5a), 2.142.35 (m, 1 H, H-5b), 2.45 (t, J = 7.4 Hz, 2 H,CH2-2 ), 4.264.34 (m, 1 H, H-4), 4.374.43 (m, 1 H, H-3), 4.50 (d,J = 4.3 Hz, 2 H, CH 2-1), 5.10 (dddd, J = 4.8 and 9.7 Hz, 1 H, H-2),6.51 (br. s, 1 H, OH), 8.47 (d, J = 8.5 Hz, 1 H, NH).


N-[(2S ,3S ,4R )-1,3,4-Trihydroxyoctadecan-2-yl]decanamide(PCER6)1 H-NMR (300 MHz, pyridine): = 0.820.95 (m, 6 H, CH 3 ),

1.151.52 (m, 30 H, CH 2 ), 1.642.04 (m, 7 H, CH 2 ), 2.202.32 (m,1 H, CH2 ), 2.27 (t, J = 7.5 Hz, 2 H, CH 2 ), 2.54 (t, J = 7.4 Hz, 2 H,CH2 ), 4.264.34 (m, 1 H, H-4), 4.374.43 (m, 1 H, H-3), 4.484.52(m, 2 H, CH 2-1), 5.10 (dddd, J = 4.8 and 9.3 Hz, 1 H, H-2), 8.47 (d,J = 8.5 Hz, 1 H, NH).


N-(1,3,4-Trihydroxyoctadecan-2-yl)dodecanamide (PCER7)1 H-NMR (300 MHz, pyridine): = 0.810.96 (m, 6 H, CH 3 ),

1.141.56 (m, 38 H, CH 2 ), 1.642.05 (m, 5 H, CH 2 , H-5a), 2.202.33 (m, 1 H, H-5b), 2.47 (t, J = 7.4 Hz, 2 H, CH 2-2 ), 4.254.35 (m,1 H, H-4), 4.374.45 (m, 1 H, H-3), 4.474.57 (m, 2 H, CH 2-1), 5.11(dddd, J = 4.8 and 9.3 Hz, 1 H, H-2), 6.18 (br. s, 1 H, OH), 6.51 (br.s, 1 H, OH), 6.60 (br. s, 1 H, OH), 8.48 (d, J = 8.4 Hz, 1 H, NH).


N-(1,3,4-Trihydroxyoctadecan-2-yl)tetradecanamide(PCER8)1 H-NMR (300 MHz, pyridine, 50 C): = 0.92 (app. t, J = 6.6

Hz, 6 H, 2 CH 3 ), 1.221.57 (m, 42 H, CH 2 ), 1.642.02 (m, 5 H,CH2 , H-5a), 2.162.31 (m, 1 H, H-5b), 2.46 (t, J = 7.4 Hz, 2 H,CH2-6), 4.204.38 (m, 2 H, H-4 and H-3), 4.394.52 (m, 2 H,

CH2-1), 5.02 (dddd, J = 4.7 and 9.3 Hz, 1 H, H-2), 5.86 (br. s, 1 H,OH), 6.19 (d, J = 4.8 Hz, 1 H, OH), 6.32 (br. s, 1 H, OH), 8.15 (d,J = 8.3 Hz, 1 H, NH).


N-(1,3,4-Trihydroxyoctadecan-2-yl)benzamide (PCER9)1 H-NMR (300 MHz, pyridine): = 0.830.96 (m, 3 H, CH 3 ),

1.141.51 (m, 22 H, CH 2 ), 1.581.81 (m, 1 H, H-6a), 1.852.07 (m,2 H, H-5a, H-6b), 2.222.37 (m, 1 H, H-5b), 4.264.43 (m, 1 H,H-4), 4.474.55 (t, J = 5.1 Hz, 1 H, H-3), 4.59 (app. d, J = 5.1 Hz,2 H, CH2-1), 5.29 (dddd, J = 5.0 and 10.1 Hz, 1 H, H-2), 6.28 (br.s, 1 H, OH), 6.69 (br. s, 1 H, OH), 7.317.45 (m, 5 H, ar. H), 8.19(d, J = 7.1 Hz, 1 H, NH).


Synthesis of Azone (1-Dodecylazacycloheptan-2-One)Substitution of 1-bromododecane by -caprolactam gave ac-

cess to azone ( fig. 2). To a solution of -caprolactam (250 mg, 2.2mmol) in toluene (6 ml) at 0 C, sodium hydride (110 mg, 2.65mmol) was added, and the mixture was stirred for 30 min at thistemperature. 1-Bromododecane (0.64 ml, 2.65 mmol) was added,and the reaction mixture was heated to reflux for 20 h. MeOH(3 ml) was added to quench the reaction, and the solvents wereremoved under reduced pressure. The residue was taken into di-ethyl ether and washed with brine, the organic layer was dried overmagnesium sulphate, filtered and evaporated to dryness. Purifica-tion by column chromatography (hexane:ethyl acetate 8: 2) gavethe title compound (629 mg, quantitative) as a colourless oil.

1 H-NMR (300 MHz, deuterated chloroform): = 0.80 (t, J = 6.8Hz, 3 H, CH 3 ), 1.101.28 (m, 19 H, CH 2 ), 1.351.47 (m, 2 H, CH 2 ),1.501.70 (m, 6 H, CH 2 ), 2.392.46 (m, 2 H, CH 2 ), 3.203.30 (m,4 H, CH2 ).

Exact mass (ESI-MS) for C18 H36 NO [M + H] + found, 282.2802;calcd, 282.2791.

Preparations of Franz Diffusion Dose SolutionsDose solutions were prepared at 80% of the maximal solubility

in a 50/50 ethanol/H 2 O (% V/V) solution of caffeine, ibuprofenand testosterone to obtain the same thermodynamic activity forthe 3 transdermal model compounds. The solubility at 32 C (i.e.the temperature of the skin) in the 50/50 ethanol/H 2 O solutions(mean SE, n = 3) amounts to 67.31 7.01, 9.71 0.12 and 53.80 3.00 mg/ml for caffeine, testosterone and ibuprofen, respective-ly. In these dose solutions, the PCERs were added to obtain a finalconcentration of 1% (W/V). The dose solutions were kept at 32 0.5 C until they were applied on the skin. Azone (1% W/V) wasincluded in the experiments as a positive control, while negative

NH

O

N

O C12 H 25

Fig. 2. Synthesis of azone. Reagents and conditions: (i) sodium hy-dride, toluene, 0 C, 30 min; (ii) 1-bromododecane, reflux, 20 h.


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controls were included as well (dose solutions without penetrationenhancer). Azone and some PCERs were not tested as penetrationenhancer on ibuprofen, as no effects were observed in the first ex-periments with selected PCERs in combination with ibuprofen.

In vitro Permeation StudyStatic FDCs (Logan Instruments Corp., N.J., USA) with a recep-

tor compartment of 5 ml and an available diffusion area of 0.64 cm2 were used to determine the skin permeation of the 3 model com-pounds in the different dose formulations. Human skin was used,and the analyses were minimally done in duplicate using a random-ized blocked design. The skin samples were obtained after a cos-metic reduction surgery from 7 healthy female patients (52 6 yearsold, mean SD) which were supplied by the Department of Plasticand Reconstructive Surgery of the University Hospital (Ghent, Bel-gium). Confidentiality procedures with informed consent were ap-plied. Immediately after the surgical procedure, the skin was cleanedwith 0.01M PBS, pH 7.4, the subcutaneous fat was removed and theskin was subsequently stored at 20 C for not longer than 6 months.Just before the start of the FDC experiments, the full-thickness skinwas thawed, mounted on a template and sliced to obtain a skin thick-ness of approximately 400 m, using an electrical powered derma-tome. An actual skin thickness of 340 11 m (mean SE, n = 70),399 9 m (n = 40), 414 11 m (n = 20), 259 5 (n = 40), 269 8 (n = 20), 317 13 m (n = 40) and 403 23 m (n = 20) was ex-perimentally determined with a micrometer (Mitutoyo, Tokyo, Ja-pan) from the different patients. The receptor chambers were filledwith 0.01M PBS for caffeine and ibuprofen and with 5% W/V BSAin PBS for testosterone. The skin samples were visually inspected forskin damage and were mounted on the FDC between the donor andthe receptor chambers, with the epidermis side upwards ensuringthat no air was present under the skin. A Teflon-coated magneticstirring bar (400 r.p.m.) allowed that the receptor fluid was continu-ously mixed. Skin integrity was controlled by measuring the skinimpedance using an automatic microprocessor-controlled TinsleyLCR Impedance Bridge (Croydon, UK). Skin pieces displaying animpedance value below 10 k were discarded and replaced by a newskin piece [17]. 500 l of the dose solutions were applied on the skinsurface with a micropipette. The donor chamber was covered withparafilm to prevent evaporation of the dose formulations. Duringthe FDC experiment, the temperature of the receptor compartmentwas kept constant at 32 C by a water jacket. 200-l samples of recep-tor fluid were taken at regular time intervals (0, 1, 2, 4, 8, 12, 18, 21and 24 h) from the sample port and were immediately replaced by200 l fresh receptor fluid. This was taken into account for the cal-culation of the cumulative permeated concentrations. Immediatelyafter the last sample had been drawn, the remaining dose formula-tion was removed from the skin surfaces using a cotton swab. Theepidermis and dermis were separated with forceps, and the modelcompounds were extracted from the skin layers with ethanol to con-struct a mass balance and to obtain the ratios

24 h 24 hepidermis dermis24 h 24 h vehiculum epidermis

C Cand .

C C

The concentration of the model compounds in the (epi)dermiswas obtained by dividing the amount of extracted model com-pound (experimentally determined) by the volume of the (epi)der-mis [thickness of (epi)dermis (cm) skin surface (0.64 cm 2 )]. Anepidermis thickness of 50 m was always taken, and the thickness

of the dermis was calculated from the total skin thickness minusthe thickness of the epidermis. The concentration of the modelcompounds in the remaining dose solution after 24 h was calcu-lated by dividing the amount of the dose solution left after 24 h(experimentally determined) by the applied volume of the dosesolution.

A linear relationship of the individual cumulative amount ofthe model compounds versus time was observed. All individualruns confirmed the steady state as the model compounds were re-leased at a constant rate into the receptor fluid. Sink conditionswere also achieved: the receptor fluid did not contain more than10% of the saturated concentration of testosterone (adsorbed toBSA), ibuprofen (fully ionized in PBS) and caffeine (water solubil-ity of 29 mg/ml at 32 C) after 24 h.

Sample PreparationBefore HPLC-UV analysis, the BSA present in the 200 l sam-

ples with testosterone was precipitated with 200 l acetonitrile.The tubes were vortexed for 10 s, and after 10 min of incubation atroom temperature, the tubes were centrifuged for 10 min (14,243 g ). The clear supernatant was used for HPLC-UV analysis.

High-Performance Liquid ChromatographyThe model compounds in the different samples taken from th

FDC experiment were analysed using a validated high-throughpHPLC-UV method. A Waters Alliance 2695 separation module aa dual absorbance detector 2487 equipped with Empower 2 softw(Waters) were part of the HPLC apparatus. 25 l of each sample injected on a Symmetry C18 column (75 mm 4.6 mm, 3.5 m par-ticle size; Waters) with an appropriate guard column. The sampcompartment was kept constant at 20 C, while the column teperature was maintained at 30 C. A degassed isocratic mobile phconsisting of a preset composition of: 0.1% FA in H2 O (A) and 0.1%FA in MeOH (B) at a flow rate of 1.0 ml/min was used. A mobphase composition of 75/25 A/B (% V/V) was used for the deternation of caffeine and ibuprofen, while a 30/70 A/B (% V/V) coposition was used for the testosterone determination. UV detectiwas performed at 272, 254 and 220 nm for caffeine, testosterone ibuprofen, respectively, with peak areas used for quantification [.

Calculation of Skin Permeation ParametersThe cumulative amounts of the model components (in micro-

grams) permeated through human skin were plotted as a functionof time (in hours). For the calculations of the transdermal param-eters, the individual graphs were used. Steady-state flux [J ss , g/(cm2 h)] was calculated from the slope of the linear portion of thecumulative amount versus time curve divided by 0.64 to correctfor the exposed skin area (in square centimetres). The lag time (inhours) was obtained by setting y = 0 in the individual linear regres-sion equation. The Q 1d is the cumulative quantity, expressed aspercentage of the effective dose applied, obtained after 1 day. Theindicated parameters are the secondary parameters. The primaryparameters are calculated in accordance with ECETOC, CEFIC[18]; the permeability coefficient (K p , cm/h) was calculated as fol-lows:

,ss pd

J K

C

where Cd (g/ml) is the concentration of the model compoundin the dose formulation. Furthermore, the apparent diffusion


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(Dm , cm2/h) and partition (K m ) coefficients were determined usingthe following equations:

2

6

,

mlag

pm

m

d D

t

K d K

D

where d and t lag are the measured skin thickness (cm) and the lagtime (h), respectively. The transdermal penetration-enhancing ra-tio (ER) was calculated as follows:

,

,

. p with penetration enhancer

p without penetration enhancer

K ER

K

Results

Penetration-Enhancing Effect of PCERs on CaffeineA linear relationship of all individual cumulative

amounts of caffeine versus time was observed. The meancumulative amounts versus time curves for caffeine with

1% of the different PCERs are visually presented in fig-ure 3. Two groups of PCERs are visible in the graph:PCER4, 5 and 710 with cumulative amounts of caffeinebetween 130 and 250 g after 24 h and another group(PCER13 and 6) with cumulative amounts between340 and 410 g. The curves of this latter group exhibithigher slopes than the curves of the first group. Thehighest cumulative amount of caffeine after 24 h wasobtained in the presence of the positive control azone

(1,609 g). For the PCERs, the highest cumulativeamount was obtained with PCER1 (phytosphingosine),as observed in figure 3 , and PCER2, PCER3 and PCER6also provided an enhanced cumulative amount of caf-feine compared to the control.

All Jss , lag time and Q 1d data can be found in the onlinesupplementary table 1 (for all online suppl. material, seewww.karger.com/doi/10.1159/000365730). The skin flux of caffeine (mean SE, n = 10) without the PCERs was11.27 1.84 g/cm 2 h. Neither PCER8 nor PCER9 hadany effect on this value, and the flux values (mean SE,n = 2) were determined as 11.53 2.50 and 10.89 1.26g/cm2 h, respectively. These values contrast the muchhigher flux values in the presence of PCER1, PCER2 andPCER3 of 35.44 14.98 (n = 4), 29.26 8.94 (n = 3) and31.98 13.97 g/cm 2 h (n = 4), respectively.

The percentage of caffeine of the applied dose solutionpenetrated through the skin after 24 h (mean SE) forazone is 5.98 1.97% (n = 4) and for PCER1, 2 and 3 1.60 0.76 (n = 4), 1.45 0.47 (n = 3) and 1.44 0.73%(n = 4). The overall apparent permeability coefficients

(Kp ), as well as the derived diffusion (D m ) and partition(Km ) coefficients for caffeine are given in table 2 . The ex-perimentally determined K p of the negative control, i.e.caffeine without any PCER (2.11 104 cm/h), is compa-rable with our previous result (2.23 10 4 cm/h) [15] . Thehighest Kp values of all the PCERs were seen with PCER2,PCER3 and PCER6. The enhanced permeability of caf-feine in the presence of PCERs mainly originated fromthe partitioning of caffeine out of the dose solution into

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CF + PCER7

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CF + PCER4

CF + PCER1

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CF + PCER9

CF + PCER2

CF + PCER6

CF +

CF +

CF + PCER10CF + PCER8 Fig. 3. Cumulative amount versus timecurve of caffeine (CF) with 1% (W/V) ofthe PCERs (mean SE, n = 210).

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the skin, as evidenced by the overall increased K m values,as well as the ratio

24 hepidermis

24 h vehiculum

C

.C

The higher the ratio24 hepidermis24 h vehiculum

C

C.

the higher the concentration of caffeine in the epider-mis, indicating the increased partition of caffeine fromthe dose solution into the epidermis.

The distribution of caffeine in the dose solution, thedifferent skin layers and the receptor fluid is graphicallyrepresented in figure 4 . The concentration of caffeine was

overall higher in the remaining dose solution than in theepidermis. The concentrations in the dermis were ap-proximately 10 times lower than in the epidermis. Thereceptor fluid concentration of caffeine after 24 h wasmore than 100 times lower than in the epidermis.

PCER1, PCER2, PCER3 and PCER6 exhibited pene-tration ERs for caffeine of 2.48, 2.75, 2.62 and 2.70, re-spectively, which are shown in table 3 . The PCERs withan ER higher than 2 correspond to the upper group of

Table 2. Primary transdermal parameters of caffeine (CF; mean SE, n = 2 10)

Dose solution Kp, 10 4 cm/h D m, 10 5 cm2/h Km, 10 1 24 hepidermis24 h vehiculum

C

C

24 hdermis

24 hepidermis

CC

CF 2.11 0.35 5.27 1.22 1.68 0.34 0.36 0.11CF + azone 25.50 7.93 2.91 0.74 27.71 6.72 0.77 0.07CF + PCER1 7.02 2.97 2.72 1.06 8.31 1.79 0.70 0.04CF + PCER2 5.73 1.72 10.31 5.43 3.44 1.43 1.40 0.02CF + PCER3 5.98 2.63 27.32 25.20 4.38 2.10 0.49 0.04

CF + PCER4 3.09

0.25 5.03

2.41 2.93

1.40 0.43 0.03CF + PCER5 3.86 1.59 3.13 1.05 4.03 1.37 0.39 0.19CF + PCER6 5.36 0.98 3.90 1.75 5.55 0.91 0.86 0.03CF + PCER7 2.49 0.63 7.54 4.22 1.77 0.67 0.34 0.04CF + PCER8 2.18 0.50 4.49 1.03 1.92 0.71 0.14 0.05CF + PCER9 2.05 0.25 4.94 0.90 1.58 0.35 0.10 0.05CF + PCER10 3.51 0.75 10.93 5.28 1.42 0.53 0.58 0.02

CF

CF + PCER2

CF + PCER3

10.0

8.0

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Remainingdose

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CF + PCER4

CF + PCER5

CF + PCER6

CF + PCER7

CF + PCER8

CF + PCER9

CF + PCER10

CF + PCER1

CF + AZ Fig. 4. Concentration of caffeine (CF; mean SE, n = 210) in different compartments24 h after dermal application in FDC. AZ =Azone.

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PCERs in the graph presented in figure 3 . Azone had thehighest ER for caffeine, namely 11.40.

Penetration-Enhancing Effect of PCERs onTestosteroneFor testosterone, a linear relationship of all individual

cumulative amounts versus time was observed. Figure 5shows the mean amounts of testosterone accumulated inthe receptor fluid as a function of time for all the investi-gated PCER penetration enhancers. Azone provided thehighest cumulative amount for testosterone after 24 h(127 g). As can be observed, PCER2 and PCER10 re-

sulted in the highest accumulation of testosterone in thereceptor compartment after 24 h. On the other hand,PCER7, PCER8 and PCER9 did not modify to a signifi-cant extent the amount of permeated testosterone withrespect to that observed with the control. Three groups ofPCERs are observed in figure 5 : PCERs giving the highestcumulative amount of testosterone after 24 h (82104g), PCERs with a moderate (57 g) and PCERs with thelowest cumulative amount of testosterone (2637 g).

The secondary transdermal parameters for testoster-one are given in supplementary table 1. The testosteroneflux (mean SE, n = 9) without penetration enhancer was2.95 0.48 g/cm2 h, while the Jss values (mean SE,n = 4) for testosterone with PCER2 and PCER10 were9.06 3.22 and 9.10 1.23 g/cm2 h, respectively.

The permeability, diffusion and partitioning coeffi-cients for testosterone are given in table 4 . The experi-mentally determined K p of the negative control (3.92 104 cm/h) is almost the same as our previous result (3.97 104 cm/h) [15] . There is no clear trend noticeable be-

tween high partition coefficient values of testosteroneand the ratio24 hepidermis24 h vehiculum

C

C.

Therefore, in contrast to caffeine, we cannot unambigu-ously conclude that high concentrations of testosteronein the epidermis can be explained by a high partitioningof testosterone from the dose solution into the epidermis.

Table 3. ERs for caffeine (CF, n = 2 4), testosterone (TES, n = 1 4) and ibuprofen (IBU, n = 2) (mean SE)

Penetrationenhancer

ER CF ER TES ER IBU

Azone 11.40 3.30 4.13 1.32 PCER1 2.48 0.44 2.08 0.56 0.98 0.31PCER2 2.75 0.74 2.56 0.13 0.92 0.03PCER3 2.62 0.93 3.48 1.19 0.09

PCER4 1.39 0.05 2.53 PCER5 1.81 0.59 2.04 0.14 PCER6 2.70 0.45 2.05 0.48 1.25 0.21PCER7 1.13 0.11 1.08 PCER8 0.96 0.11 1.12 0.11 1.42 0.05PCER9 0.92 0.01 1.19 0.16 PCER10 1.55 0.16 4.84 0.79

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TES + PCER3

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TES + PCER8 ES + PCER9

TES + PCER2

TES + PCER6

TES +

TES +

ES + PCER10 Fig. 5. Cumulative amount versus timecurve of testosterone (TES) with 1% (W/V)of the PCERs (mean SE, n = 29).

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Figure 6 presents the distribution of testosterone intothe dose solution, the different skin layers and the recep-tor fluid. The concentration of testosterone remained

higher in the epidermis than in the remaining dose solu-tion: the epidermis functioned as a skin reservoir for tes-tosterone, the concentration gradient decreased towardsthe dermis and receptor fluid.

The enhancing ratios of the various PCERs for testos-terone are given in table 3 . A remarkable ER of 4.8 is ob-served for PCER10, followed by an ER of 4.1 for azoneand 3.5 for PCER3, while PCER1, PCER2, PCER4,PCER5 and PCER6 have a moderate penetration-en-

hancing effect between 2 and 3 for testosterone throughthe skin. The other PCERs with an ER value lower than2, belonging to the lowest group in figure 5 , have no

pharmaceutically relevant effect on the permeability oftestosterone.

Penetration-Enhancing Effect of PCERs on IbuprofenAll individual cumulative amounts of ibuprofen ver-

sus time showed a linear relationship. The curves of themean cumulative amounts of ibuprofen in the receptorfluid as a function of time for all PCERs assayed are illus-trated in figure 7 .

TES

TES + PCER2

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TES + AZ

Remainingdose

Epidermis Dermis 10 Receptorfluid 100

Fig. 6. Concentration of testosterone (TES;mean SE, n = 29) in different compart-ments 24 h after dermal application inFDC. AZ = Azone.

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Table 4. Primary transdermal parameters of testosterone (TES; mean SE, n = 2 9)

Dose solution K p, 10 4 cm/h Dm, 10 5 cm2/h Km, 10 1 24 hepidermis24 h vehiculum

C

C

24 hdermis

24 hepidermis

CC

TES 3.92 0.621 6.40 2.08 2.84 0.50 5.46 0.07TES + azone 12.70 2.47 36.90 30.91 12.99 7.10 7.32 0.05TES + PCER1 11.02 0.39 4.28 0.24 9.73 0.72 0.87 0.10TES + PCER2 12.03 4.10 2.26 0.77 17.01 5.26 0.68 0.14TES + PCER3 11.91 0.71 3.27 0.53 13.56 0.31 5.33 0.02TES + PCER4 9.72 1.57 4.70 1.06 7.71 0.12 0.66 0.08TES + PCER5 6.81 0.97 3.21 0.69 6.98 0.95 3.01 0.21TES + PCER6 11.37 5.04 2.88 0.59 11.43 4.06 7.20 0.04TES + PCER7 3.27 0.23 7.07 1.20 1.75 0.32 4.42 0.03TES + PCER8 2.94 0.45 5.43 2.20 2.32 0.63 9.15 0.05TES + PCER9 3.74 0.45 19.55 14.82 2.36 0.84 3.71 0.05TES + PCER10 12.49 1.77 2.74 0.61 15.66 3.49 2.36 0.12


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The secondary transdermal parameters for ibuprofencan be found in the online supplementary table 1. Theflux (mean SE, n = 4) of ibuprofen without the PCERswas 82.41 6.95 g/cm2 h, while in the presence ofPCER8, PCER3 and PCER6 the flux (mean SE, n = 2)was 99.65 14.94, 84.35 9.29 and 87.67 0.72 g/cm2 h,respectively. Besides, the other investigated PCERs didnot result in a significant increase in the ibuprofen fluxthrough the skin.

The primary transdermal parameters for ibuprofen(Kp , Dm , Km ) are given in table 5 . The experimentally de-termined K p of the negative control (2.05 10 3 cm/h) iscomparable with our previous result (2.19 10 3 cm/h)[15]. The distribution of ibuprofen into the different skinlayers is given in figure 8. The highest concentration ofibuprofen after 24 h was located in the remaining dosesolution. A decrease in ibuprofen concentration is ob-served towards the dermis and the receptor fluid.

The ERs of the PCERs are presented in table 3 , but nopenetration-enhancing effect was observed for ibuprofen.All ratios are below the value of 2.

Discussion

The shielding function of the human SC executesphysicochemical limitations to the nature of molecules

which can be passed through the barrier lipid matrix.Hence, it is important to use an appropriate penetrationenhancer in the development of transdermal drug deliv-ery systems to increase the skin permeation rate (flux) ofpermeants without significantly affecting toxicity and/orharming the skin [10, 1921] .

The most widely explored enhancement strategy in volves the use of lipid-protein-partitioning chemicals thdecrease the skin barrier properties and subsequently allo

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Fig. 7. Cumulative amount versus timecurve of ibuprofen (IBU) with 1% (W/V) ofthe PCERs (mean SE, n = 24).

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Table 5. Primary transdermal parameters of ibuprofen (IBU; mean SE, n = 2 4)

Dose solution K p, 10 4 cm/h D m, 10 5 cm2/h Km, 10 1 24 hepidermis24 h vehiculum

C

C

24 hdermis

24 hepidermis

CC

IBU 20.53 1.81 175.58 114.82 1.17 0.53 0.61 0.16IBU + PCER1 21.38 6.82 31.25 0.52 2.50 0.54 0.62 0.53IBU + PCER2 20.00 0.60 56.87 51.44 6.52 5.73 0.22 0.36IBU + PCER3 22.43 2.97 95.40 49.42 1.07 0.37 0.81 0.13IBU + PCER6 23.17 1.09 94.74 16.32 0.91 0.04 0.88 0.10IBU + PCER8 27.06 4.76 158.20 66.13 0.86 0.52 0.43 0.13


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the entry of even poorly penetrating molecules into thedeeper layers. It appears that interactions with the SC inter-cellular lipids between the corneocytes is the most effectiveone [9, 22]. The lipid interaction can occur near the polarhead and between the hydrophobic tail of the lipid bilayer[23]. An example of such a penetration enhancer is the li-pophilic molecule azone, which was the first chemical de-signed as a transdermal penetration enhancer. Azone parti-tions into the lipids of the SC and disrupts the lipid bilayerstructure, producing a more fluid environment, by pre- venting chain crystallization. In addition, azone reduces thediffusional resistance of the skin. Drugs could pass the skinmore efficiently through this less rigid environment. Theefficacy of azone is dependent on the used concentration: itappears to be most effective at low concentrations (0.15%)and is often used in concentrations between 1 and 3% [20,24, 25]. PCERs are amphiphilic molecules which penetratewith their hydrophobic part near the lipid fatty acid chainsand with their hydrophilic part near the polar head of thelipids, indicating that they disrupt the lipid packing and in-

duce a lateral fluidization of the lamellae [23].The optimal chain length for chemical penetration en-hancers derived from amino acids was found to be 1012carbons [9]. This chain length causes disorder between themuch longer hydrophobic chains of ceramides present inthe SC, leading to easier drug permeation. These generalfindings are in contrast with studies about ceramides aspenetration enhancers [13]. In the latter study, ceramideswith a sphingosine chain length of 18 carbons and fatty

acid chains with various carbon atoms were investigateand it seemed that the optimal acyl chain length for thpenetration behaviour was between 4 and 6 carbons. the current study, we investigated PCERs, featuringcommon 18-carbon sphingoid base and different acchain lengths. The investigated PCERs exerted a compound-dependent penetration-enhancing effect. SomPCERs enhanced the penetration of caffeine and testostone, but lack an enhancing effect on ibuprofen. PCERwith an ER of more than 2 having an effect on caffeinewell as on testosterone are PCER1, PCER2, PCER3 aPCER6. The PCERs containing a fatty acid chain with(PCER3) and 10 (PCER6) carbons are in correspondenwith the previous findings for ceramide-like compoundreported in the literature [9, 13]. However, in contrast towhat was found for earlier reported ceramides, we oserved that PCERs with different acyl chain lengths aexhibited a penetration-enhancing effect, indicating ththe extra OH group on C4 of the sphingoid base may playa role as well. Our study confirms the ceramide findin

that there is no linear relationship between the acyl chalength and the skin permeability [13]. Caffeine is a hydrophilic compound with a logP val

of 1.06 (calculated using HyperChem). The overall obs vation that the highest concentration of caffeine was fouin the remaining dose solution seems logical, as the epidmis, with the SC as outer layer, is a more lipophilic sklayer. Caffeine will thus hardly diffuse to and penetrate inthis skin layer, remaining in the hydrophilic dose solutio

Fig. 8. Concentration of ibuprofen (IBU;mean SE, n = 24) in different compart-ments 24 h after dermal application inFDC.

IBU

45.0

40.0

35.0

30.0

25.0

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References 1 Bouwstra JA, Gooris GS, Ponec M: The lipidorganization of the skin barrier: liquid andcrystalline domains coexist in lamellar phas-es. J Biol Phys 2002;28: 211223.

2 Bouwstra JA, Honeywell-Nguyena PL,Goorisa GS, Ponec M: Structure of the skinbarrier and its modulation by vesicular for-mulations. Prog Lipid Res 2003; 42: 136.

3 Mizutani Y, Mitsutake S, Tsuji K, Kihara A,Igarashi Y: Ceramide biosynthesis in kerati-nocyte and its role in skin function. Biochi-mie 2009; 91: 784790.

4 Geilen CC, Wieder T, Orfanos CE: Ceramidesignalling: regulatory role in cell proliferation,differentiation and apoptosis in human epi-dermis. Arch Dermatol Res 1997; 289: 559566.

5 Goi FM, Alonso A: Biophysics of sphingo-lipids. I. Membrane properties of sphingo-sine, ceramides and other simple sphingolip-ids. Biochim Biophys Acta 2006;1758: 19021921.

6 Masukawa Y, Narita H, Shimizu E, Kondo N,Sugai Y, Oba T, Homma R, Ishikawa J, Taka-gi Y, Kitahara T, Takema Y, Kita K: Charac-terization of overall ceramide species in hu-man stratum corneum. J Lipid Res 2008; 49:14661476.

7 Van Smeden J, Hoppel L, Van der Heijden R,Hankemeier T, Vreeken RJ, Bouwstra JA: LC/MS analysis of stratum corneum lipids: ce-ramide profiling and discovery. J Lipid Res2011; 52: 12111221.

The highest permeability (K p ) was observed for PCERswith 2 (PCER2) and 10 (PCER6) carbons in the acyl chain.Co-administration of caffeine with PCER3 (4 carbons)and, strikingly, also PCER1 (phytosphingosine) gave abiomedically significant penetration-enhancing effect(ratio of more than 2).

In contrast to caffeine, the highest amount of testoster-one after 24 h was found in the epidermis. Testosteroneis a more lipophilic compound with a logP of 3.84 (calcu-lated using HyperChem) and prefers to partition in theepidermis, out of the dose solution. On the other hand,the skin layer following the epidermis is the more hydro-philic dermis. Hence, testosterone will concentrate in theepidermis, and will penetrate with difficulty from the epi-dermis into the dermis.

Co-administration with the PCER containing a 2-OH-phenyl functional group (PCER10) resulted in the highestpenetration-enhancing effect on testosterone, whilePCER10 did not have a biomedically significant enhanc-ing effect on caffeine. Phytosphingosine (PCER1) and theother PCERs having an acyl chain with 210 carbons hadalso a penetration-enhancing effect on testosterone withan ER of more than 2, while with PCER7, PCER8 andPCER9, containing 12 carbons, 14 carbons and a phenylgroup, respectively, no significant enhancing effect oc-curred.

Ibuprofen, with a logP of 3.83 (calculated using Hyper-Chem), contains also an acidic carboxyl function with apKa between 4.4 and 5.7, depending on the ethanol con-centration of the dose solution [26] . At the skin surface,ibuprofen will thus be partly ionized. Nevertheless, thereis still an appreciable flux of ibuprofen through the skin.After 24 h, the highest concentration of ibuprofen wasoverall found in the remaining dose solution, different

from testosterone with a similar logP, but no ionizablegroup. Nevertheless, none of the investigated PCERs hada penetration-enhancing effect on ibuprofen, similar tothe situation with spilanthol as penetration enhancer.

Conclusion

The investigated PCERs exhibited a compound-de-pendent penetration-enhancing effect on caffeine andtestosterone but not on ibuprofen. In combination withcaffeine, PCER1 (phytosphingosine), PCER2 (2 carbons),PCER3 (4 carbons) and PCER6 (10 carbons) exhibited anER of 2.48, 2.75, 2.62 and 2.70, respectively. PCER1PCER6 and PCER10 (hydroxylated benzene function)gave an ER of more than 2 for testosterone, with PCER10showing a remarkable ER of 4.84. None of the investi-gated PCERs modified the transdermal behaviour of ibu-profen. This study indicates that selected PCERs are po-tentially useful in transdermal formulations to facilitatetransport of the active ingredient through the SC.

Acknowledgement

The authors like to thank the Special Research Fund of GhentUniversity (BOF 01D23812 to Lien Taevernier) for their funding,as well as the Institute for the Promotion of Innovation throughScience and Technology in Flanders (IWT-Vlaanderen) (No.091257 to Jente Boonen).

Disclosure Statement

No competing financial interests exist.


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8 Vvrov K, Hrablek A, Dolezal P, Holas T,Zbytovsk J: L -Serine and glycine based ce-ramide analogues as transdermal permeationenhancers: polar head size and hydrogenbonding. Bioorg Med Chem Lett 2003; 13:23512353.

9 Vvrov K, Hrablek A, Dolezal P, Samalova L,Palt K, Zbytovsk J, Holas T, Klimentov J:Synthetic ceramide analogues as skin perme-ation enhancers: structure-activity relation-ships. Bioorg Med Chem 2003; 11: 53815390.

10 Vvrov K, Zbytovsk J, Hrablek A: Amphi-philic transdermal permeation enhancers:structure-activity relationships. Curr MedChem 2005; 12: 22732291.

11 Novotn J, Janov B, Novotn M, HrablekA, Vvrov K: Short-chain ceramides de-crease skin barrier properties. Skin Pharma-col Physiol 2009; 22: 2230.

12 Sinko B, Kokosi J, Avdeef A, Takacs-Novak K:A PAMPA study of the permeability-enhanc-ing effect of new ceramide analogues. ChemBiodivers 2009; 6: 18671874.

13 Janov B, Zbytovsk J, Lorenc P, Vavrysov

H, Palt K, Hrablek A, Vvrov K: Effect ofceramide acyl chain length on skin permeabil-ity and thermotropic phase behavior of mod-el stratum corneum lipid membranes. Bio-chim Biophys Acta 2001; 1811: 129137.

14 Baert B, Deconinck E, Van Gele M, SlodickaM, Stoppie P, Bod S, Slegers G, Vander Hey-den Y, Lambert J, Beetens J, De Spiegeleer B:Transdermal penetration behaviour of drugs:CART-clustering, QSPR and selection ofmodel compounds. Bioorg Med Chem 2007;15: 69436955.

15 De Spiegeleer B, Boonen J, Malysheva SV,Diana Di Mavungu J, De Saeger S, Roche N,Blondeel P, Taevernier L, Veryser L: Skin pen-etration enhancing properties of the plant N -alkylamide spilanthol. J Ethnopharmacol2013; 148: 117125.

16 Veryser L, Boonen J, Mehuys E, Roche N, Re-mon JP, Peremans K, Burvenich C, De Spie-geleer B: Transdermal evaluation of caffeinein different formulations and excipients. JCaffeine Res 2013; 3: 4146.

17 De Spiegeleer B, Baert B, Vergote V, Van Dor-pe S: Development of system suitability testsfor in vitro skin integrity control: impedanceand capacitance. 11th Int Perspect PercutanPenetration Conf, La Grande Motte, March2008.

18 ECETOC, Monograph Report No 20: Percu-taneous Absorption. Brussels, European Cen-tre for Ecotoxicology and Toxicology ofChemicals, 1993.

19 Barry BW: Novel mechanisms and devices toenable successful transdermal drug delivery.Eur J Pharm Sci 2001; 14: 101114.

20 Williams AC, Barry BW: Penetration enhanc-ers. Adv Drug Delivery Rev 2004; 56: 603618.

21 Guillard EC, Tfayli A, Laugel C, Baillat-GuffroA: Molecular interactions of penetration en-hancers within ceramides organization: a FTIRapproach. Eur J Pharm Sci 2009; 36: 192199.

22 Zadymova NM: Colloidochemical aspects oftransdermal drug delivery (review). Colloid J2013; 5: 491503.

23 Jampilek J, Brychtova K: Azone analogues: classification, design, and transdermal penetrationprinciples. Med Res Rev 2012; 32: 907947.

24 Barry BW: Mode of action of penetration en-hancers in human skin. J Control Release1987; 6: 8597.

25 Lane ME: Skin penetration enhancers. Int JPharm 2013; 447: 1221.

26 Watkinson RM: Influence on the solubility,ionization and permeation characteristics ofibuprofen in silicone and human skin. SkinPharmacol Physiol 2009; 22: 1521.

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