Journal of Controlled Release 165 (2013) 91–100

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Journal of Controlled Release

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Amino acid derivatives as transdermal permeation enhancers

Barbora Janůšová, Barbora Školová, Katarína Tükörová, Lea Wojnarová, Tomáš Šimůnek, Přemysl Mladěnka,Tomáš Filipský, Michal Říha, Jaroslav Roh, Karel Palát, Alexandr Hrabálek, Kateřina Vávrová ⁎Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Czech Republic

Abbreviations: Ala, alanine; Azone, N-dodecylazep(dimethylaminopropanoate); DDAK, dodecyl 6-(dimethylment ratio; Gly, glycine; HC, hydrocortisone; IR, infrared;PG, propylene glycol; Pro, proline; Sar, sarcosine; SC, stratu12; TEWL, transepidermal water loss; TH, theophylline.⁎ Corresponding author at: Charles University in Prague

Králové, Heyrovského 1203, 500 05 Hradec Králové, Czec497; fax: +420 495 067 166.

E-mail address: katerina.vavrova@faf.cuni.cz (K. Váv

0168-3659/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.jconrel.2012.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 July 2012Accepted 3 November 2012Available online 12 November 2012

Keywords:Transdermal drug deliveryin vitro/in vivo skin absorptionPenetration enhancerAmino acidStratum corneum

Transdermal permeation enhancers are compounds that temporarily decrease skin barrier properties to pro-mote drug flux. In this study, we investigated enhancers with amino acids (proline, sarcosine, alanine,β-alanine, and glycine) attached to hydrophobic chain(s) via a biodegradable ester link. The double-chainlipid-like substances displayed no enhancing effect, whereas single-chain substances significantly increasedskin permeability. The proline derivative L-Pro2 reached enhancement ratios of up to 40 at 1% concentration,which is higher than that of thewell-established and standard enhancers Azone, DDAIP, DDAK, and Transkarbam12. No stereoselectivity was observed. L-Pro2 acted synergistically with propylene glycol. Infrared studiesrevealed that L-Pro2 forms a separate liquid ordered phase in the stratum corneum lipids and has no significanteffect on proteins. L-Pro2 action was at least partially reversible as measured by skin electrical impedance.Toxicity in keratinocyte (HaCaT) andfibroblast (3T3) cell lines showed IC50 values ranging from tens to hundredsof μM, which is comparable with standard enhancers. Furthermore, L-Pro2 was rapidly decomposed in plasma.In vivo transdermal absorption studies in rats confirmed the enhancing activity of L-Pro2 and suggested its neg-ligible skin toxicity and minimal effect on transepidermal water loss. These properties make L-Pro2 a promisingcandidate for potential clinical use.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Transdermal drug delivery offers several advantages over conven-tional routes of administration, such as avoidance of the first-passmetabolism, stable plasma levels, lower incidence of side effects, andimproved patient compliance. However, due to the remarkable barrierproperties of the skin's uppermost layer, the stratum corneum (SC),transdermal administration has not yet achieved its full potential. Oneapproach to enabling this route of administration for a wider range ofdrugs is the use of chemical compounds that temporarily increasedrug flux, known as permeation enhancers or penetration/absorptionpromoters (for reviews, see refs. [1–6]). Although much effort hasgone into the development of these compounds, their wider use inclinical practice is hampered by the fact that their mechanisms of actionand their potential toxicity are still not fully understood.

Already in the 1980s, many surfactant-like compounds with C10–C12 chain length have been identified as potent permeation enhancers

an-2-one; DDAIP, dodecyl 2-amino)hexanoate; ER, enhance-PBS, phosphate-buffered saline;m corneum; T12, Transkarbam

, Faculty of Pharmacy in Hradech Republic. Tel.: +420 495 067

rová).

rights reserved.

[7–14]; for reviews, see refs. [1,6,15]. Most of these enhancers, however,affect also viable epidermal cells provoking significant skin irritation.One of the rare exceptions to this rule is an alanine derivative dodecyl2-(dimethylamino)propanoate (DDAIP, NexAct, [16]), probably becauseof its biodegradability by epidermal esterases. To identify more en-hancers or their combinations with high potency and low irritation risk,Mitragotri's group developed a high-throughput screening tool basedon the effect of enhancer on the skin electrical properties [17–20]. Theydemonstrated that there exist classes of enhancers for which potencyand irritation are not particularlywell related [17]. One of the compoundswhich displayed apparent efficacy without noticeable irritation potentialwas another amino acid derivative, N-lauroylsarcosine [21,22].

Thus, amino-acid derivatives seem to be among the most promisingclass of permeation enhancers, especially those with a hydrophobic“tail” attached to an amino acid “head” via a biodegradable linkage,e.g. an ester bond (Fig. 1A). This molecular design is advantageousdue to the amphiphilic structure of such enhancer, which could allowit to incorporate into the SC lipid barrier and disrupt the tight arrange-ment of the membrane lipids. Then, after reaching enzymatically activenucleated epidermis, its labile bond could be hydrolyzed, thus releasingknown non-toxic compounds with much lower irritation potential.This approach to designing permeation enhancers resulted in the iden-tification of highly potent enhancers with favorable properties, suchas DDAIP [16], Transkarbam 12 (T12, [23,24]), tranexamic acid deriva-tives [25], and dodecyl 6-(dimethylamino)hexanoate (DDAK, [26–28],Fig. 1B).

Fig. 1. Schematic representation of the design principles of the amino acid permeation enhancers (panel A), enhancers used as positive standards in this work (panel B), synthesis(panel C), and structures of the studied amino acid permeation enhancers (panel D). Reagents and conditions: a — dodecanol, HCl, 120 °C, 7 h; b — dodecanoic acid,dicyclohexylcarbodiimide, 4-dimethylaminopyridine, CHCl3, rt, 20 h; c — acetic anhydride, 4-dimethylaminopyridine, CHCl3, rt, 5 h; d — ethylbromide, triethylamine, tetrahydro-furan, rt, 8 h. R1 and R2=H, CH3, –(CH2)3–(Pro).

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Here, we explore the use of the amino acids glycine (Gly), L- andD-alanine (L-Ala and D-Ala), β-alanine (β-Ala), sarcosine (Sar), andL- and D-proline (L-Pro and D-Pro) as headgroup components ofpermeation enhancers (Fig. 1D). Our interest in α-amino acids wasoriginally based on L-serine, a starting amino acid in the biosynthesisof the key skin barrier lipids, ceramides. We hypothesized thatenhancers and ceramides must bear a certain structural similarity toensure the molecular interaction required for their enhancing effect.Thus, in a previous study, we attached two hydrophobic tails tothis amino acid to mimic the ceramide structure. We found that thechain length was crucial: L-serine with 12C chains behaved as a mod-erate permeation enhancer [29,30] while its homolog 14S24, with thesame chain lengths as in ceramides, was able to repair skin barrierperturbed by various insults [31,32]. The replacement of L-Ser byGly, i.e., removal of the hydroxymethyl group, increased its enhancingactivity, probably due to its lower ability to form hydrogen bonds[29,30].

In this study, we prepared and studied a series of double-chainenhancers based on the Gly homolog β-Ala, its isomers L-Ala andSar, and also on the conformationally restricted cyclic amino acidL-Pro. The latter two amino acids were included to test our hypothesisthat hydrogen bonding ability negatively influences the enhancingactivity, and because Pro [33,34] and Sar [35] derivatives were previ-ously reported to elicit permeation-enhancing activity. Interestingly,Gly, β-Ala, and Pro were also used to prepare prodrugs of 5-OH-DPAT for transdermal iontophoretic delivery [36].

We also prepared a series of single-chain enhancers based onthe same amino acids to confirm our previous suggestion that theremoval of one long hydrophobic tail increases enhancing activity.The effects of the prepared amino acid derivatives were comparedwith known standard enhancers including Azone [37], DDAIP,DDAK, and T12 (Fig. 1B). We also studied the reversibility of the effectof L-Pro2, the best enhancer of this group, by electrical impedancemeasurements, and its interaction with the skin barrier lipids andproteins by infrared spectroscopy. For this purpose, L-Pro2–D25

with perdeuterated alkyl chain was synthesized. The toxicities ofselected enhancers and the possible involvement of apoptosis wereassessed in keratinocyte HaCaT and fibroblast 3T3 cell cultures andcompared to known enhancers. Furthermore, L- and D-enantiomersof selected enhancers were evaluated to address any potential stereo-selective action/toxicity. The most potent enhancer, L-Pro2, was alsostudied in vivo in rats to confirm its enhancing properties, toxicity,effect on transepidermal water loss (TEWL) and biodegradability.

2. Materials and methods

2.1. Synthesis of enhancers

The synthetic procedures and properties of theprepared compoundsincluding deuterated L-Pro2–D25 are given in the Supplementary data.

2.2. Donor samples for permeation studies

Control donor samples were prepared as 5% (w/v) suspensionsof theophylline (TH) or 2% (w/v) suspensions of hydrocortisone(HC) in distilled water, 60% propylene glycol (PG, v/v), and isopropylmyristate, respectively. TH (mol. weight 180 g/mol, logP ~0) and HC(362 g/mol, logP 1.6) were selected as model permeability markersrepresenting drugs of different physicochemical properties. Enhancersamples for co-application experiments were prepared by adding1% (w/v) of the studied enhancer to the aforementioned drug sus-pensions. The samples were stirred at 50 °C for 5 min and thenallowed to equilibrate at 37 °C for 24 h. Before application to theskin, the samples were resuspended. The concentrations were select-ed so that all samples were saturated with both the pertinent modeldrug and studied enhancer to maintain the same thermodynamicactivity throughout the experiments. To determine whether theadded enhancers had any effects on the solubility of the drugs inthe donor solvent, the samples were prepared in triplicate as de-scribed above and allowed to equilibrate. After 24 h, the suspensions

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were centrifuged at 6,700 ×g for 5 min; the supernatant was with-drawn, diluted with the pertinent mobile phase and analyzed byHPLC. L-Pro2 donor samples for impedance and IR experimentscontaining 1% (w/v) L-Pro2 in 60% PG without the model drug wereprepared likewise. Moreover, 1% enhancer dispersions in water and60% PG without the drugs were also prepared to check their solubilityand stability at 37 °C.

2.3. Permeation experiments

Skin permeabilitywas evaluated usingmodified Franz diffusion cellswith an available diffusion area of 1 cm2 and an acceptor volume of ap-proximately 17 ml. Frozen porcine skin (for details, see Supplementarydata) was slowly thawed, cut into pieces of 2×2 cm, mounted intothe diffusion cells dermal side down and sealed with silicone grease.The acceptor compartment was filled with phosphate-buffered saline(PBS, containing 10 mM phosphate buffer, 137 mM NaCl and 2.7 mMKCl) at pH 7.4 with 0.03% of sodium azide as a preservative, and thevolume of the acceptor phase was measured for each cell and includedin the calculation. The Franz diffusion cells with mounted skin sampleswere placed in a water bath with a constant temperature of 32 °Cequipped with a magnetic stirrer. After an equilibration period of 1 h,skin integrity was checked bymeasurement of the electrical impedance(see later) and then 200 μl (i.e., an infinite dose) of the donor samplewas applied to the SC side of the skin and covered with a glass slide.The acceptor phase was stirred at 32 °C throughout the experiment.Sink conditions were maintained for all drugs. Samples of the acceptorphase (0.6 ml) were withdrawn at predetermined time intervals andreplaced with fresh buffer solution. The permeation experiments wererun for 48 h and 52 h for TH and HC, respectively, to reach the pseudosteady-state to calculate the drug flux. TH and HC were determined byHPLC as described previously [24]; for details, see Supplementarydata. The cumulative amount of the drug permeated across the skin,corrected for the acceptor phase replacement, was plotted againsttime, and the steady state flux was calculated from the linear regionof the plot. The enhancement ratio (ER) was calculated as a ratio ofthe flux with and without the enhancer.

2.4. Skin electrical impedance

The skin integrity before each permeation experiment and thereversibility of the skin barrier function after L-Pro2 treatment wasstudied by measuring the transdermal electrical impedance using anLCR meter 4080 (Conrad electronic, Hirschau, Germany, measuringrange 20 Ω–10 MΩ, error at kΩ valuesb0.5%) operated in a parallelmode with an alternating frequency of 120 Hz, parameters thatyield the best sensitivity to small impedance changes [38]. The skinsamples were mounted into the Franz diffusion cells, the acceptorcompartments were filled with PBS at pH 7.4, and the cells wereequilibrated at 32 °C for 1 h as described above. Half a milliliter ofPBS was introduced into a donor compartment and the baselineskin resistance (kΩ×cm2) was measured by two stainless steel elec-trodes carefully immersed into PBS in the donor and acceptor com-partments of the diffusion cell. The buffer solution was removedfrom the donor compartment using a cotton swab, and 200 μl of thedonor sample containing 1% (w/v) of L-Pro2 in 60% PG was applied.The first set of control cells received 200 μl of 60% PG without theenhancer, and the second set received 200 μl of distilled water todistinguish the effect of hydration from that of PG and L-Pro2. Thedonor samples were removed after 2 h or 48 h and the skin surfacewas washed twice with 0.5 ml of PBS and gently blotted dry. Theimpedance was measured for 20 h or 63 h. The reported values arenormalized to the baseline value (i.e., the impedance before treat-ment) of each skin fragment.

2.5. Isolation of SC and SC lipids

The SC sheets were isolated by trypsin treatment [39] and thelipids were extracted using a modified Bligh and Dyer method [40].For details, see Supplementary data.

2.6. Infrared (IR) spectroscopy

Before the experiment, SC sheets were cut into small pieces(ca. 1 mg) and treated with 50 μl of distilled water (control), 60%PG, or 1% L-Pro2 in 60% PG at 32 °C. Isolated SC lipids were eitheruntreated (control), or treated with 20 μl of 60% PG, 1% L-Pro2 in60% PG or 1% L-Pro2–D25 in 60% PG at 32 °C. After 2 h, the excess so-lution was carefully removed and the samples were examined by IRspectroscopy. Fourier transform IR spectra of the samples werecollected on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific,USA) equipped with a single-reflection MIRacle attenuated totalreflectance (ATR) germanium crystal. A clamping mechanism with aconstant clamping pressure was used for all experiments. The spectrawere generated by co-addition of 128 scans collected at 4 cm−1 res-olution. The spectra were analyzed using the Bruker OPUS software.The exact peak positions were determined from second derivativespectra and by peak fitting if needed.

2.7. Cell lines

The HaCaT spontaneously immortalized human keratinocyte cellline was purchased from the Cell Lines Service (Eppelheim, Germany)and the 3T3-Swiss albino mouse embryonic fibroblast cell line wasfrom the American Type Culture Collection (ATCC, distributed byLGC Standards, Poland). Cells were cultured in Dulbecco's modifiedEagle's medium (DMEM, Lonza, Belgium) supplemented with 10%heat-inactivated fetal bovine serum (Lonza), 1% penicillin/streptomycinsolution (Lonza) and 10 mMHEPES buffer (Sigma, Germany) in 75 cm2

tissue culture flasks (TPP, Switzerland) at 37 °C in a humidified atmo-sphere of 5% CO2. Sub-confluent cells were subcultured every3–4 days. For cytotoxicity experiments and caspase activity determina-tions, cells were seeded in 96-well plates (TPP) at a density of 5000 cellsper well. For morphology and fluorescence assessments, cells wereseeded at a density of 75,000 cells per well in 12-well plates (TPP)24 h prior to the addition of the test drugs. The cells plus test substanceswere then incubated for 48 h under standard conditions. WhereasDDAK and DDAIP (both in the form of hydrochlorides) were dissolvedin PBS, dimethylsulfoxide was used to dissolve Azone, and ethanolwas used for T12, Sar2, L-Pro2, and D-Pro2. The corresponding concen-trations of solvents were always present in the control incubationmedia.

2.8. Cellular toxicities of selected enhancers

Cellular toxicities were determined by the ability of activemitochondria to change yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-difenyltetrazolium bromide (MTT; Sigma) to purple formazan.After the 48-h experimental incubations, 25 μl of MTT solution inPBS (3 mg/ml) was added to 100 μl medium in each well. After incu-bation for 2 h at 37 °C, the cells were lysed with 0.1 M hydrochloricacid in isopropanol with 10% Triton X-100. Plates were vigorouslyshaken for 3 h to fully dissolve the formazan crystals. The optical den-sities were measured at λ=570 nm, subtracting the λ=690 nmbackground using a Tecan Infinite 200 Mplate reader (Tecan, Austria).The viabilities of experimental groups were expressed as percentagesof untreated controls (100%).

Changes in cellular morphology were evaluated using an invertedepifluorescence microscope (Eclipse TS100, Nikon, Japan) equippedwith a digital cooled camera (1300Q, VDS Vosskühler, Germany)and software NIS-Elements AR 2.30 (Laboratory Imaging, Czech

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Republic). Cellular death was visualized using double staining ofnuclei with Hoechst 33342 (Molecular Probes) and propidium iodide(PI; Molecular Probes), which are well established as sensitive proce-dures for determining apoptosis and necrosis. Hoechst 33342 is ablue-fluorescent probe (λex=360 nm; λem=460 nm) that stains allnuclei. In apoptotic cells, chromatin condensation occurs and apopto-tic cells can thus be identified as those with condensed and moreintensely stained chromatin. The red DNA-binding dye, PI (λex=560 nm; λem=630 nm), is unable to cross the plasma membrane ofliving cells, but readily enters necrotic (or late-stage apoptotic) cellsand stains their nuclei red. Cells were loaded with 3 μg/ml of Hoechst33342 and 10 μg/ml of PI for 15 min at room temperature and sampleimages were taken using the microscope set-up described above.

2.9. Caspase activity

To assess the possible involvement of apoptosis in cell killing,the activities of basic initiator (apical) as well as effector (executive)caspases were determined after 48-h incubations of cells with select-ed concentrations of permeation enhancers. The cells were lysed byadding 100 μl of lysis buffer (100 mM HEPES, 10 mM CHAPS,10 mM DTT, pH 7.4) to 100 μl medium in each well. Lysates were im-mediately frozen at −80 °C. Thawed lysates were then used forcaspase activity assessments using luminescent kits for caspases 3/7,8 and 9 (Promega, U.S.A.). The caspase activities of experimentalgroups were corrected for the protein content in each sample (BCAkit, Sigma) andwere expressed as a percentage of activities of untreatedcontrols (100%). The luminescence wasmeasured using a Tecan Infinite200 M plate reader.

2.10. In vivo transdermal permeation, TEWL and dermal toxicity

Wistar:Han female rats (Biotest s.r.o., Czech Republic) were housedin cages in an air-conditioned room with a periodic 12-h light–darkcycles for two weeks. During this period, the rats had free access totap water and standard pellet diet for rodents. Before the experiments,they were fasted overnight. The study was performed under thesupervision of the Ethical Committee of Charles University in Prague,Faculty of Pharmacy in Hradec Králové and conformed to The Guidefor the Care andUse of LaboratoryAnimals published by theUSNationalInstitutes of Health (NIH Publication No. 85-23, revised 1996).

The rats were anaesthetized with 1.05 g/kg urethane i.p., hair onthe back was removed using an electric clipper and the formulations(1.2 ml/kg of 5% TH in 60% PG with or without 1% L-Pro2) wereapplied to 4 cm2 gauze patch, which was held in place with non-irritating occlusive patch. Blood samples were collected from the leftcommon iliac artery each 30 min for 8 h. TH in plasma samples wasdetermined by HPLC using protein precipitation with methanol andcaffeine as an internal standard (see Supplementary data).

For TEWL and toxicity measurements, rats received a) no treat-ment, b) 60% PG, c) 1% L-Pro2 in 60% PG, or d) 5% L-Pro2 in 60% PG(all at 400 μl in a 4 cm2 gauze patch) for 6 h. After this period, thepatches were removed; the surface of the skin briefly washed withethanol to remove the residual formulations, blotted dry and carefullyinspected for any erythema/edema/dryness. After 30 min, TEWL wasrecorded using Tewameter® TM 300 (Courage+Khazaka, Germany).Animals were killed by intravenous administration of 1 ml of 1 M KCland skin biopsies were collected from the sites exposed to the testedformulations. The effect of L-Pro2 on skin viability was determined bya TTC assay (see Supplementary data).

2.11. Stability of L-Pro2 in rat plasma and PBS at 37 °C

Rat plasma or PBS at pH 7.4 (1 ml, n=4) containing L-Pro2 at50 μg/ml were incubated at 37 °C. At predetermined time intervals,10 μl samples were withdrawn and vigorously mixed with 90 μl of

methanol. The PBS samples were analyzed directly, plasma sampleswere centrifuged at 6700 ×g for 5 min; the supernatant was with-drawn and then analyzed by HPLC (see Supplementary data).

2.12. Statistical analysis

A two-tailed Student's t-test was used when comparing twodifferent conditions. When comparing three or more conditions, aone-way analysis of variance (ANOVA) with a Bonferroni post-hoctest was performed using SigmaStat for Windows 3.5 (SPSS, U.S.A.).In all cases, pb0.05 was considered significant. The concentrationsof enhancers inducing a 50% decrease in viability (IC50) were calcu-lated using the CalcuSyn 2.0 software (Biosoft, Cambridge, U.K.).Data are presented as means±SEM and the number of replicates(n) is given in the pertinent figures.

3. Results

3.1. Synthesis

The target compounds were designed as amino acid dodecyl estershaving either another 12C chain (referred to as double-chainenhancers) or a short acetyl or ethyl (single-chain enhancers) at itsamino group. To simplify their synthesis, the common fragments,i.e., the dodecyl esters, were synthesized first and used for the pre-paration of both enhancer series; the amino group was acylatedby carbodiimide coupling or using acetic anhydride, or alkylated byethylbromide (Fig. 1C and D). All products were crystalline, exceptfor the Pro derivatives. The logP values ranged from 4.9 to 6.8 in thesingle chain enhancers and 10.4–12.1 in the double chain compounds.At 1% concentration, all enhancers were saturated in water and 60%PG (the solubilities were less than or equal to 0.58%), and stable for atleast 48 h.

3.2. Single-chain amino acid derivatives are better enhancers thandouble-chain derivatives

The flux values of the model drug TH through the skin with andwithout the studied amino acid derivatives and standard enhancersare presented in Fig. 2. Panel A shows the ability of the studiedenhancers to increase the flux of TH when dispersed in a simpleaqueous vehicle. Without an enhancer, the flux of TH was 4.04±0.41 μg/cm2/h. Azone, which is used as a standard for permeation-enhancing activity [37], increased this value 2.7-fold under theseconditions, DDAIP was inactive, while enhancers DDAK and T12 en-hanced TH flux up to 7-fold. A non-significant increase in flux (similarto that shown by Azone), was elicited by all the studied double-chainenhancers. The single-chain enhancers were generally more effectivethan their double-chain counterparts (except for β-Ala2), with themost active compound being L-Pro2, giving a TH flux of 27.9±3.1 μg/cm2/h and a corresponding ER value of 6.9. L-Pro2 was signifi-cantly more active than Azone and DDAIP, and comparable to DDAKand T12. The second best enhancer was Sar2 with an ER value of6.2, followed by Gly2 (ER=3.9) and L-Ala2 (ER=3.6). The solubilityof TH in the aqueous donor sample was 8.6±0.2 mg/ml. Thedouble-chain enhancers increased its solubility by 10%. It meansthat the rather small insignificant increase in flux of TH in the pres-ence of the double-chain substances was caused by the increasedsolubility of TH in the donor vehicle. The single-chain enhancershad no effect on TH solubility in the donor vehicle, suggesting a differ-ent mechanism of action. We also tested some of the intermediates,i.e., amino acid dodecyl esters with a free amino group, but no sig-nificant effects were observed (data not shown).

Fig. 2. The effects of the prepared amino acid permeation enhancers (1%) on the trans-dermal flux of a model drug TH (5%) dispersed in water (panel A) and in 60% PG (panelB), respectively. Control represents the flux of TH without an enhancer. Mean±SEM,n≥4 for the double-chain enhancers and≥8 for the single-chain ones; * indicatesstatistically significant difference compared to the respective control or as indicatedat pb0.05.

Fig. 3. The effects of L- and D-enantiomers of Pro12, Pro2, ProEt, and Ala2 permeationenhancers (1%) on the transdermal flux of a model drug TH (5%) dispersed in water(panel A) and in 60% PG (panel B). Control represents the flux of THwithout an enhancer.Mean±SEM, n≥4 for the double-chain enhancers and≥8 for the single-chain ones; * in-dicates statistically significant difference compared to the respective control at pb0.05.

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3.3. The most active permeation enhancer L-Pro2 acts synergisticallywith PG

The enhancers were then combined with PG in an attemptto increase their activity because (co)solvents like PG or ethanoloften act synergistically with amphiphilic surfactant-like enhancers[8,12,13,41,42]. In 60% PG, the flux of TH without enhancers was1.78±0.48 μg/cm2/h, which was slightly lower than that in a simpleaqueous vehicle (Fig. 2, panel B). However, when PG was combinedwith the most active enhancer L-Pro2, the TH flux value increased to70.3±7.7 μg/cm2/h, which is 40 times higher than that of PG aloneand 2.5 times higher than that of L-Pro2 dispersed in water. Suchsynergy was not observed for the other studied enhancers, forwhich the TH flux values were similar when applied in water or PG.None of the positive controls, i.e., known enhancers reached the activ-ity of L-Pro2. DDAIP, DDAK, and T12 reached ER values of 7.8, 23, and19, respectively — thus, L-Pro2 was significantly more efficient thanall of them. The solubility of TH in 60% PG was 25±2 mg/ml; noneof the tested enhancers altered this value significantly suggestingtheir direct action in SC. We also investigated a lipophilic isopropylmyristate as a donor vehicle. However, none of the prepared en-hancers was able to increase the TH flux (2.63±0.63 μg/cm2/h)significantly under these conditions (data not shown).

3.4. Substitution of N-acetyl by N-ethyl in Pro-derived enhancersdecreases enhancing activity

We also prepared and evaluated Pro derivatives in which theN-acetyl was substituted with an ethyl group to mimic the basic ter-tiary amino group in the highly active enhancers DDAK and DDAIP.However, this change led to a significant decrease in activity, almostto values comparable with the double-chain enhancers (Fig. 3).

3.5. The skin permeation-enhancing activity of Pro and Ala derivativesis not stereoselective

As several of the studied amino acid enhancers are chiral, we exam-ined whether their interaction with chiral SC components includingceramides or proteins differs between enantiomers. Thus, we alsoprepared the unnatural D-enantiomers of the selected enhancers,namely D-Pro2, D-Pro12, D-ProEt, and D-Ala2. However, no significantdifference was observed between the L- and D-enantiomers, eitherdouble- or single-chain (Fig. 3).

3.6. L-Pro2 significantly increases skin permeability for both lipophilicand hydrophilic permeability markers

To further examine the ability of L-Pro2 to improve skin permeabilityfor a broader range of potential drugs, the flux of HC, a relatively largelipophilic neutral molecule, was studied. The flux of HC in 60% PGthrough the skin was 0.14±0.09 μg/cm2/h. Combining PG with 1%L-Pro2 increased the HC flux 47 times to 6.54±0.87 μg/cm2/h; theskin permeation profile is shown in Fig. 4A. The solubility of HC inthe donor sample was 8.9±0.3 mg/ml; L-Pro2 increased this value1.3-fold. That means that a part of the enhancing activity of L-Pro2towards HC permeation was caused by an indirect increase ofthe drug solubility in the donor vehicle. In terms of the permeabilitycoefficients Kp, which are independent of donor concentration,L-Pro2 increased Kp value 31 times (from 1.80×10−5 cm/h to 5.65×10−4 cm/h).

Furthermore, skin electrical impedance was selected to probe theability of L-Pro2 to enhance the skin permeation of hydrophilicpermeants and to show that its action has a relatively rapid onset. Thebaseline impedance values varied between 7.5 and 26.8 kΩ×cm2.After 2 h and 48 h L-Pro2 treatment, the skin impedance reached 2.0–7.9 kΩ×cm2 and 1.5–1.9 kΩ×cm2, respectively (i.e., 35% and 8% ofthe impedance before treatment), which was significantly lower thanfor PG alone (4.2–14.9 kΩ×cm2, i.e., 74% of the baseline after 2 h and4.2–7.7 kΩ×cm2, i.e., 28% of the baseline after 48 h PG treatment,Fig. 4B).

3.7. L-Pro2 enhancement is reversible

Measurement of electrical impedance was further used to studythe ability of the skin permeability to recover from the action ofL-Pro2 enhancer. Each sample (water, 60% PG, and 1% L-Pro2 in 60%PG) was applied to the skin for 2 h or 48 h, removed, and the skin im-pedance measured for 20 h or 63 h (Fig. 5). PG-treated skin served asa control to distinguish between the actions of PG and L-Pro2; anothercontrol received only water for 2 h in order to distinguish between

Fig. 4. The effects of L-Pro2 in 60% PG on the skin permeation of HC (2%) (panel A) andskin electrical impedance after 2 and 48 h treatment (panel B). Mean±SEM, n≥4; * indi-cates statistically significant difference compared to the respective control (i.e., withoutL-Pro2) at pb0.05.

Fig. 6. The effects of L-Pro2 on the protein (panels A–C) and lipid (panels D–F) compo-nents of SC studied using IR spectroscopy. A and B— wavenumbers of amide I bands ataround1650 and 1620 cm−1, corresponding toα-helix andβ-sheet protein conformation

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the action of PG and hydration. Fig. 5 shows that the PG alone had nosignificant effect on skin impedance and that the observed decreaseis fully attributable to the hydration of the skin [43]. Nevertheless,L-Pro2 in PG significantly decreased skin impedance already after2-h application. After L-Pro2 had been removed from the skin surface,the impedance further decreased, plateaued, and 6 h after the endof the treatment, began to rise, reaching significant recovery at 20 h(Fig. 5A). Similar trend was observed after 48-h application ofL-Pro2 (Fig. 5B).

in SC, respectively; C — relative area of α-helical conformation in SC proteins; D and E —

wavenumbers and bandwidths, respectively, of symmetric methylene stretching of iso-lated SC lipids; F — wavenumbers of symmetric CD2 stretching of L-Pro2–D25, eitherneat or in the SC lipids. Mean±SEM, n≥6; *Statistically significant difference comparedto the respective control or as indicated at pb0.05.

3.8. L-Pro2 forms separated liquid ordered phase in the SC lipids; PGchanges conformation of SC proteins

The mechanisms of action of L-Pro2 and PG in the skin barrierwere studied using IR spectroscopy. First, isolated SC sheets wereexamined. Upon treatment with the enhancers, significant changeswere found in the protein regions, in particular in the amide I vibra-tions, which are mostly composed of amide carbonyl stretching, andare sensitive to changes in protein conformation. Untreated SC dis-played a strong amide I vibration at around 1650 cm−1 and a weakone at 1620 cm−1 typical of α-helix and β-sheet protein conformation,respectively. The prevailingα-helix was consistent with previous stud-ies on SC proteins [44]. Treatment with PG and L-Pro2 in PG for 2 hresulted in a shift of both vibrations towards higher wavenumbers(Fig. 6A–B), suggesting a partial change in hydrogen bonding ofthe amide oxygens. The most prominent change in the amide regionwas the decrease in the relative area of the α-helix band from

Fig. 5. The reversibility of L-Pro2 effects on skin electrical impedance when applied for2 h (A) and 48 h (B). The data are expressed as % of the baseline value at time 0.Mean±SEM, n=4–10; *Statistically significant differences compared to the respectivecontrol, i.e., PG-treated skin at pb0.05, + indicates statistically significant differencesat indicated time intervals at pb0.05.

approximately 67% to 48% (Fig. 6C). All these effects on the SC proteinswere caused by PG rather than L-Pro2.

For a more detailed investigation of the effects of PG and L-Pro2on skin barrier lipids, isolated SC lipids were used to exclude the con-tribution of amino acid side chain vibrations in the C–H stretchingregion (Fig. 6D–E). L-Pro2 was found to incorporate into the SC lipidsas reflected by an increase in the area of C–H stretching bands (notshown). Such enhancer incorporation caused an increase in wave-numbers of both symmetric and asymmetric methylene stretchingfrom 2848.9 cm−1 to 2850.2 cm−1, and 2916.4 cm−1 to 2918.4 cm−1,respectively, and peak broadening by 2.0 cm−1 and 8.3 cm−1,respectively.

For a more precise interpretation of these results, we synthesizedL-Pro2–D25 with perdeuterated alkyl chain to distinguish betweenthe methylene vibrations originating from the SC lipids and theenhancer [45,46]. Incorporation of L-Pro2–D25 did not increase theSC lipid chain disorder suggesting that this enhancer forms a separatephase within the SC lipids (Fig. 6D–E). Examination of the CD2

stretching bands of neat enhancer and the SC lipids that had beenexposed to 1% L-Pro2–D25 in 60% PG for 2 h revealed these separateenhancer domains exist in a liquid ordered phase (Fig. 6F). This wasassigned according to literature data on CD2 vibrations [47–49].

3.9. Toxicities of selected enhancers in HaCaT and 3T3 cell lines arecomparable to known enhancers

The toxicity experiments were run for 48 h, since most of thestudied enhancers did not induce a 50% reduction in cell viabilityafter 24 h. Following the 48-h incubations with HaCaT keratinocyteor 3T3 fibroblast cell lines, all examined permeation enhancersinduced dose-dependent reductions in cellular viability. The IC50values — i.e., the concentration of drugs inducing a 50% decrease in

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viability — of all agents were lower in HaCaT cells (indicating highertoxicity) as compared to the 3T3 cell line (Fig. 7, panel B). In bothcell lines, T12 was the most toxic agent, with IC50 values of ~20 μM.The toxicity of the most active enhancer L-Pro2 was approximatelytwo-fold higher than that of Azone, but similar to a widely usedenhancer DDAIP and also to DDAK. No significant difference in toxic-ity was observed between the L- and D-enantiomers. The changes incellular morphology were followed by epifluorescence microscopy(Fig. 7A). Following the 48-h incubations, peripheral membraneblebbing occurred in a dose-dependent manner followed by the lossof cell shape and rounding up of cells. Furthermore, severe nuclearcondensations occurred and eventually complete loss of cellularviability was followed by the formation of cell debris. Although in in-dividual permeation enhancers these changes occurred at differentconcentrations, no conspicuous qualitative differences were observedamong the examined compounds.

We also examined the possible involvement of apoptosis by deter-mining the effects of selected enhancers (at concentrations inducingpartial toxicity) on caspase activity. In HaCaT cells (Fig. 7C), relativelyslight (approximately two-fold) but significant activation of allcaspases was observed with DDAK and T12; caspases 3/7 were alsoactivated by Azone. In 3T3 cells (Fig. 7D), a significant increase incaspase activity was observed with T12 and Pro derivatives. WhereasT12 increased caspase activity approximately two-fold, comparable tothe effect seen with an equitoxic T12 dose in HaCaT cells, the effectsof L-Pro2 and D-Pro2 were particularly pronounced, reaching≈1400–1800% of the control cells. All three caspases generally reachedcomparable levels with all assayed permeation enhancers.

3.10. L-Pro2 enhances transdermal permeation of TH in rats in vivo withnegligible effects on TEWL and skin cell viability

To examine the permeation-enhancing effect of L-Pro2 in vivo,plasma concentrations of TH after transdermal administration of 5%TH in 60% PG with or without 1% L-Pro2 to rats were monitored

Fig. 7. Toxicity of selected permeation enhancers in HaCaT keratinocyte and 3T3 fibroblast cethe activity of caspases 3/7, 8, and 9 in HaCaT (C) and 3T3 cells (D). Mean±SEM, n≥4; *St

(Fig. 8A). Without the enhancer, the plasma concentrations of THwere below 0.1 μg/ml. With 1% L-Pro2, the plasma levels of THbegan to rise after 4 h and reached 1.27±0.09 μg/ml after 8 hadministration.

Theoretically, an enhancer should increase drug flux into the bodywithout simultaneously increasing the loss of water and endogenoussubstances from the body [3]. To test this assumption, TEWL [50]was measured in vivo at the skin sites exposed to this enhancer for6 h, i.e., under the conditions leading to significant drug permeation.The TEWL value was increased up to 1.7-fold after the application ofthe tested formulations, but no significant differences were observedbetween 60% PG and L-Pro2 in PG, even when it was applied at 5times higher concentration than was needed for the enhancementeffect (Fig. 8B). Thus, this increase of water loss could be attributedmainly to PG. Furthermore, no visible changes (i.e., no erythema/edema/dryness) were observed in the rats treated with L-Pro2 ateither 1% or 5% concentration in PG. To study the dermal toxicitymore closely, cell viability was determined in skin biopsies after 6-hexposure to enhancers. Fig. 8C shows that under these conditions,the permeation enhancement effect of L-Pro2 was not accompaniedby any significant decrease of skin cell viability.

During the in vivo permeation study, no L-Pro2 was found in plas-ma suggesting either low systemic absorption or decomposition ofthis enhancer in metabolically active skin layers or plasma. To studythe biodegradability of L-Pro2, it was incubated with rat plasma andin PBS pH 7.4 at 37 °C as a control. A decomposition of L-Pro2with a half-life of ~2.5 h was found in plasma, while no significantdecrease in concentration was found in PBS (Fig. 8D).

4. Discussion

In this study, we investigated a series of transdermal permeationenhancers containing an amino acid linked to a hydrophobic chain(s)via a labile ester bond. The double-chain compounds were designedto resemble ceramides (neutral sphingolipids in the SC intercellular

ll lines. A— cellular morphology, B— IC50 values, C–D— effects of selected enhancers onatistically significant difference compared to the respective control at pb0.05.

Fig. 8. The enhancement activity of 1% L-Pro2 on transdermal delivery of TH in ratsin vivo (panel A), effects of 6-h administration of L-Pro2 (at 1% and 5%) and the vehicleitself (60% PG) on TEWL and viability of dermal cells (panels B and C, respectively) inrats in vivo, and metabolization of this enhancer in rat plasma compared to its stabilityin PBS at pH 7.4 at 37 °C (panel D). Mean±SEM, n≥3; *Statistically significant differ-ence compared to the respective control at pb0.05.

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lipid lamellae [51,52]), which are believed to be important targets ofthe enhancers [2,3]. The 12C chains were selected based on previousstudies showing that this particular length was optimal for highenhancing activity [7–14]; for reviews, see [1,6,15]. However, thesedouble-chain lipid-like compounds failed to increase skin perme-ability. On the contrary, their homologs, in which one of the two12C chains had been shortened to two carbons (referred to assingle-chain enhancers), displayed substantially increased potency.Similar results were previously found in dicarboxylic acid permeationenhancers that were also more active with only one 12C chain [53].Possible reasons for the lack of activity of the double-chain com-pounds may either be their inadequate permeability into the SClipid barrier or their similarity to ceramides, resulting in their inabil-ity to perturb the tight packing of these lipid membranes. Both possi-bilities are supported by data on ceramides showing changes in theirbehavior in lipid membranes upon shortening of the acyl chain,including their ability to translocate through the lipid lamellae andinfluence their permeability [54,55]. In fact, the only highly activepermeation enhancer having two hydrophobic chains is T12, a rela-tively unusual structure with a carbamic acid salt in its polar head.Nevertheless, the mechanism of action of T12 involves the releaseof carbon dioxide in SC, after which it continues acting as a single-chain enhancer [24]. Thus, the presence of only one 12C chainseems to be an important prerequisite for potent permeationenhancers.

In terms of polar head structure, the results of this study con-firmed the previously proposed negative effect of hydrogen bonding(especially of H-bond donors) on enhancing activity [29,30]. Whilethe enhancing potencies of Gly, Ala, and β-Ala were similar, the Sarand Pro derivatives, which are disubstituted amides, i.e., hydrogenbond acceptors only, displayed increased activity. This is consistentwith previous studies showing that Sar and Pro-based compoundsare potent permeation enhancers [33–35]. The negative role of hydro-gen bonding may be viewed in terms of higher membrane cohesion

in the polar head region in addition to hydrophobic interactionsbetween the chains. However, the substitution of amide in Pro en-hancers by a tertiary amino group resulted in a marked decrease inactivity. This is an interesting, yet unexplained finding because abasic tertiary amine is an important structural feature in DDAIP andDDAK [16,26]. This suggests that hydrogen bonding is not simply anegative factor but that an optimum level of bonding is needed forproper incorporation of an enhancer into the SC lipid lamellae, asproposed previously [53].

This study also demonstrated that the action of Pro and Ala-basedenhancers is not stereoselective. We compared the activity of severalenhancer enantiomers based on the assumption that enhancers inter-act with chiral skin barrier constituents, either ceramides or proteins,and that such interaction may be stereoselective. However, we foundno differences in the action of enhancer enantiomers, which is consis-tent with previous data on enhancers with the chiral center in thehydrophobic chain [56] or the polar head [26].

After defining the basic structure–activity relationships, we fo-cused on the best enhancer of this series, L-Pro2. This agent wasable to enhance the permeation of two model drugs with differentphysicochemical properties: TH, a relatively small compound (molec-ular weight of 180 g/mol) of medium lipophilicity (logP~0) thatis likely to cross the SC lipid lamellae by free-volume diffusion, andHC, a two-fold larger lipophilic substance (362 g/mol, logP~1.6)that also permeates by lateral diffusion [57]. In addition, a significantdecrease in skin electrical impedance was found upon the action ofL-Pro2. Skin impedance or resistance is often used as a rapid parame-ter for screening permeation enhancers [58,59] and reflects the skinpermeability for ions [60,61]. Thus, L-Pro2 influences different perme-ation pathways through the skin barrier, which gives it the opportu-nity to enhance the permeation of a relatively wide range of drugs.

Given its advantages for measuring skin permeability, we also usedthe electrical impedance method to determine what happens to skinpermeability following the removal of L-Pro2 from its surface. Theresults showed a significant increase in impedance, i.e., a decrease inpermeability, after enhancer removal, suggesting partial recovery ofthe skin barrier function. The most likely explanation of this behavioris that L-Pro2 does not persist in the skin barrier and is relatively rapidlyeliminated, probably by simple diffusion into lower epidermal layers.Similar reversibility of skin impedance was observed previously afterthe treatment with permeation enhancer [26], iontophoresis [62,63],or their combination [64].

L-Pro2 was also found to act synergistically with PG, which is ingood agreement with previous studies [8,12,13,41,42]. This may bedue to their action on different targets in the skin barrier. PG is asmall solvent molecule, previously suggested to influence the confor-mation of SC proteins [65]. Because L-Pro2 is an amphiphilic com-pound, it was expected to incorporate into the SC intercellular lipidlamellae with its polar head anchored in the polar membrane regionand the hydrophobic chain protruding into the hydrophobic core ofthe lamellae. To study this hypothesis, interactions between L-Pro2and PG and isolated SC or SC lipids were studied by IR spectroscopy.This technique has been widely used to monitor the action of en-hancers in the skin barrier [17,24,35,42,45,46,21]. It confirmed thatPG changes the hydrogen-bonding network of the SC proteins, andincreases the proportion of protein in the β-sheet conformation,probably by solvation of the peptide bonds. The contribution ofL-Pro2 to these changes was negligible, probably due to its more lipo-philic nature. This enhancer was found to incorporate into the SClipids, broadening both methylene stretching bands and shiftingthem to higher wavenumbers. This may be an indication of the lipiddisordering; however, such experiment cannot distinguish betweenthe lipid and enhancer chain. Thus, we repeated the experimentwith deuterated enhancer L-Pro2–D25. This approach did not revealany SC lipid chain fluidization but suggested that L-Pro2 acts byformation of separate liquid ordered domains within the SC lipids

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similarly to oleic acid [45,46]. Such phase separation may lead to for-mation of more permeable interfacial defects in the skin lipid barrier[46]. This proposed mechanism of L-Pro2 action is consistent with itsreversibility, because following elimination of the enhancer (i.e., itspenetration into deeper skin layers), the lipids may spontaneouslyreassemble.

We also examined the toxicity of selected enhancers in two skincell lines including keratinocytes and fibroblasts. The IC50 valuesshowed that the cellular toxicities of the studied Pro and Sar deriva-tives did not exceed that of a clinically used enhancer DDAIP. Wewere interested in the possible involvement of apoptosis in the cellu-lar toxicity of the studied enhancers. Apoptosis is the most importantform of programmed cell death and has been implicated in the cyto-toxic action of numerous xenobiotic compounds. Whereas caspase8 is the principal signaling molecule of the extrinsic (receptor-mediated) apoptotic pathway, caspase 9 is the key mediator of theintrinsic (mitochondrial) pathway. Caspases 3 and 7 are the mainexecutioner death proteases, catalyzing the specific cleavage ofmany key cellular proteins, and are activated in the apoptotic cellby both extrinsic and intrinsic pathways. In particular, caspase 3 is in-dispensable for apoptotic chromatin condensation and DNA frag-mentation. Whereas T12 increased caspase activity approximatelytwo-fold in both cell lines, L-Pro2 had no effect in the HaCaTkeratinocytes but caused a pronounced increase in caspase activityin the 3T3 fibroblasts. This clearly warrants further study, but is ofno particular concern, as the overall toxicity was lower in 3T3cells than in HaCaT (Fig. 7B). All three caspases generally reachedcomparable levels; hence, the observed proapoptotic action ofsome enhancers probably cannot be specifically attributed to anyexclusive apoptotic pathway.

All these in vitro characteristics suggested that L-Pro2 is a promis-ing transdermal permeation enhancer. Thus, we also performed aproof of principle in vivo study in rats. Although rat skin structureand permeability are different from human skin [66,67], the activityof established enhancers was found to be reasonably similar[68–70]. Indeed, our experiments demonstrated a pronounced andrelatively rapid enhancement of transdermal absorption of a modeldrug TH by 1% L-Pro2 without any significant dermal toxicity. Thisenhancement was also accompanied by moderate increase in waterloss, but this was attributed mainly to the PG vehicle. Furthermore,the validity of the design principle of this class of enhancers, i.e., bio-degradability of the ester linkage, was confirmed by a simple experi-ment in rat plasma as a representative of an enzymatically activebiological environment. In plasma, L-Pro2 was relatively rapidlydecomposed, but it was stable in PBS suggesting an enzymatic natureof this reaction. Given its undetectable concentrations in plasma at8 h and plasma half-life of 2.5 h, its systemic exposure is likely to bevery low. Thus, we expect that this enhancer, being an ester, may beenzymatically hydrolyzed producing safe compounds already inviable epidermis or early in plasma.

In conclusion, amino acid permeation enhancers, in particular theproline-based compound L-Pro2, possess an advantageous combina-tion of high activity, reversible action, and low toxicity, which makethem promising candidates for potential clinical use. The limitationsof the current study include the lack of data on long-term dermaland systemic toxicities, and enhancers' absorption, metabolizationand elimination. This ADME characterization of the most promisingenhancers clearly warrants further studies.

Acknowledgements

This work was supported by the Czech Science Foundation (pro-ject no. 207/11/0365) and Charles University (SVV 265 001). Wealso thank Hana Mikešová and Assoc. Prof. Jiří Kuneš for IR andNMR spectroscopy.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2012.11.003.

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