Rat epidermal keratinocyte organotypic culture (ROC) compared to human cadaver skin: The effect of skin permeation enhancers

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Rat epidermal keratinocyte organotypic culture (ROC) compared to human cadaver skin: The effect of skin permeation enhancers b ¨ Sari Pappinen a,∗ , Sanna Tikkinen a , Sanna Pasonen-Seppanen , c a,d a,e ¨ , Marjukka Suhonen , Arto Urtti Lasse Murtomaki a

Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland Department of Anatomy, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland c Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, Kemistintie 1, 02150 Espoo, Finland d Biopharmaceutical In Vitro Laboratory, Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland e Drug Discovery and Development Technology Center, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland b

a r t i c l e

i n f o

a b s t r a c t

Article history:

The objective of this study was to evaluate the response of the rat epidermal keratinocyte

Received 27 June 2006

organotypic culture (ROC) to permeation enhancers, and to compare these responses

Received in revised form

to those in human cadaver skin. Different concentrations of two mixtures for enhanc-

6 November 2006

ing permeation were investigated, sodium dodecyl sulfate:phenyl piperazine and methyl

Accepted 12 November 2006

pyrrolidone:dodecyl pyridinium chloride, using skin impedance spectroscopy and two

Published on line 24 November 2006

experimental compounds, the lipophilic corticosterone and the hydrophilic sucrose. The chemical irritation effects of the formulations were evaluated based on leakage of lactate

Keywords:

dehydrogenase enzyme (LDH) and cellular morphological perturbation. This study provides

Permeation enhancer

evidence for direct correlations of permeation/permeation, impedance/impedance and per-

Permeability

mation/impedance between the culture model and human skin. The only exception was

Impedance

the enhancer induced permeation of sucrose which was 1–40-fold higher in ROC compared

Irritation

to human skin, reflecting the more disordered lipid organization in stratum corneum and

Cell culture

consequently the greater number of polar pathways. LDH leakage and cellular morphol-

Keratinocyte

ogy indicated that it was possible to differentiate between safe permeation enhancers from

ROC

irritating agents. This is not only the first study to have compared the enhancer effects on

Human skin

a cultured skin model with human skin, but also it has demonstrated enhancer induced irritation using an artificial skin model. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Cultured skin alternatives have appeared during the last decade, and there is an increasing demand for good cell culture models that can be used in pharmaceutical and

∗

toxicological studies. If these models are to replace isolated samples of animal or human skin or to simulate in vivo experiments, the models need to be similar to human skin in terms of drug permeability, skin irritation, metabolic function, lipid composition, and other physiological features. Intact native

Corresponding author. Tel.: +358 17 163466; fax: +358 17 162252. E-mail address: sari.pappinen@uku.fi (S. Pappinen). Abbreviations: DPC, dodecyl pyridinium chloride; EF, enhancement factor; LDH, lactate dehydrogenase; MP, methyl pyrrolidone; PP, phenyl piperazine; REK, rat epidermal kerationocyte; ROC, REK organotypic culture; SDS, sodium dodecyl sulfate 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.11.013

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skin begins to deteriorate within a few hours after its separation from the underlying tissue. In contrast, cultured skin models can remain viable for long periods in vitro experiments, as long as culture conditions are maintained. This allows more opportunities to perform more elaborate experiments. The uppermost thin layer, stratum corneum, is the most important barrier for controlling drug permeation. The stratum corneum is a tight barrier with a complex lipid composition and a unique lipid organization. Despite its obvious importance, the permeability barrier of cultured skin models has not been carefully evaluated. Based on the few comparative reports between cell models and human skin it indicates that the skin models are significantly more permeable than human skin (Asbill et al., 2000; Wagner et al., 2001; Schmook et al., 2001; Dreher et al., 2002; Lotte et al., 2002). In these previous studies only a small group of chemicals (1–4) have been used in a single experiment. The permeabilities of the cell models were mostly 2- to 20-fold greater than intact human skin, but occasionally differences as great as 1000-fold have been observed, and the cell models were invariably more leaky than human skin. Permeation enhancers, substances that temporarily reduce the skin resistance and thereby enhance drug passage, have been used in topical formulations to maximize drug penetration. Permeation enhancers are typically studied in permeability experiments in which drug transfer across the skin is determined in diffusion chambers (Li and Higuchi, 2006). Measurement of skin electrical impedance has been suggested as an alternative method to measure skin permeability (Kontturi et al., 1993; Kalia and Guy, 1997; Karande and Mitragotri, 2002; Karande et al., 2004, 2005). The high electrical resistance of the skin is related to the impermeable structure of the stratum corneum (Yamamoto and Yamamoto, 1976; Sekkat et al., 2002). Accordingly, electrical resistance of the skin has been used to confirm skin integrity in vitro and in vivo (Kalia and Guy, 1997), to assess post-iontophoretic skin resistance recovery (Turner et al., 1997; Curdy et al., 2002; Kanebako et al., 2002a,b; Li et al., 2003; Zhu et al., 2003), to evaluate the irritation potential of chemicals (Heylings et al., 2001, 2003), and to measure the effects of permeation enhancers on skin integrity (Kontturi et al., 1993; Kalia and Guy, 1997; Karande and Mitragotri, 2002; Karande et al., 2004, 2005). Measurement of skin electrical impedance is a very rapid method compared to drug permeation experiments, and it could represent a very useful alternative to permeability studies if a direct correlation between skin electrical impedance and drug permeation could be shown. However, only Mitragotri and co-workers (Jain et al., 2006; Karande et al., 2006) have previously investigated this correlation. Practical use of chemical permeation enhancers requires careful balancing of their benefits and risks, because several potential enhancers are known to irritate skin when interfering with viable cell layers of the skin. Mixtures of enhancers are often more efficient than single chemicals (Mitragotri, 2000), and these potentially permit the use of lower, nonirritating, concentrations in the combinations. It is still not possible to predict the safety of enhancers on a theoretical basis, and therefore cultured skin models offer new possibilities to study both enhancer potency and skin irritation in the same experimental system. As far as we are aware, only

241

Godwin et al. (1997) have studied permeation enhancers in a cultured skin model, and they observed that the data obtained with the cell model did agree with the in vitro data from human skin. The rat epidermal keratinocyte organotypic culture (ROC) is a three-dimensional skin culture model. The model develops when keratinocytes are grown on a collagen gel in culture ¨ inserts at an air–liquid interface (Pasonen-Seppanen et al., 2001a,b). ROC is the first skin permeation model that has been appropriately demonstrated to resemble human epidermis in drug permeation studies: a total of 18 different solutes with lipophilicities (i.e., log P) ranging over 8 orders of magnitude were used (Suhonen et al., 2003). On average, the ROC model indicated twice as high a permeability coefficient (range 0.3–5.2) as obtained with isolated human cadaver epidermis. The ROC model was also successful in predicting the skin irritation potential of topically applied chemicals when the extent of irritation was assessed based on lactate dehydrogenase (LDH) leakage, interleukin 1␣ release and histological perturbation (Pappinen et al., 2005). These results showed a good linear correlation of LDH and IL-1␣ release with the results of official in vivo irritation tests on rabbit skin (ECETOC, 1995; Bancley et al., 1996). The purpose of the present study was to evaluate the response of the cultured skin model (ROC) to permeation enhancers, and to compare these responses to those obtained from intact human skin that has been isolated post-mortem. The differences between the binary enhancer compositions were small, which enabled us to study the enhancer effects in detail. We first tested the effects of 48 enhancer formulations on skin electrical impedance, and then 16 selected formulations were used in drug permeation studies and the results were correlated with impedance data. Finally, to confirm that the enhancer potential and the skin irritation could be tested using ROC skin culture model, the irritation potential of five chemically different formulations was investigated.

2.

Materials and methods

2.1.

Materials

Test chemicals sodium dodecyl sulfate (SDS), phenyl piperazine (PP), methyl pyrrolidone (MP) and dodecyl pyridinium chloride (DPC) were obtained from Sigma–Aldrich (Steinhem, Germany) and Fluka (Buchs, Germany). Cell culture media Minimal Essential Medium (MEM; without l-glutamine), Dulbecco’s MEM (DMEM1; with 4500 mg/l glucose), Dulbecco’s MEM (DMEM2; with 4500 mg/l glucose, without phenol red and l-glutamine), Earle’s Balanced Salt Solution (EBSS, 10×), 7.5% sodium bicarbonate solution and sterile Phosphate Buffered Saline (PBS, 10×) were obtained from Gibco BRL (Life Technologies Ltd., Paisley, Scotland). Penicillin–streptomycin sulfate, l-glutamine, trypsin–EDTA and l-ascorbic acid were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Transwell tissue culture inserts (24 mm diameter, 3.0 ␮m pore size) were obtained from Costar (Cambridge, MA, USA). Nunclon Surface 96-well plates for LDH measurement were from Nunc A/S (Roskilde, Denmark). Lactate dehydrogenase was from Fluka (Buchs,

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Switzerland). CytoTox 96® Non-Radioactive Cytotoxicity Assay for LDH measurement was from Promega (Madison, WI, USA). Histochoice tissue fixative was obtained from Amresco (Solon, OH, USA). Radiolabeled [14 C]-sucrose (588.0 mCi/mmol) and [3 H]-corticosterone (70.0 Ci/mmol) were from PerkinElmer (Boston, MA, USA).

2.2.

REK organotypic culture (ROC)

An epidermal keratinocyte (REK) cell line from newborn rats, which was originally isolated by Baden and Kubilus (1983), was used. The cells were routinely cultured in MEM (with 10% FBS, 4 mM l-glutamine, 50 U/ml penicillin and 50 ␮g/ml streptomycin sulfate) in culture flasks at 37 ◦ C, in 5% CO2 and in 95% humidity according to a protocol described earlier ¨ (Pasonen-Seppanen et al., 2001a,b). REK organotypic cultures (ROC) were prepared by seeding the REKs onto collagenated Transwell culture inserts and grown on DMEM1 (with 10% FBS, 4 mM l-glutamine, 50 U/ml penicillin and 50 ␮g/ml streptomycin sulfate) supplemented with 40 ␮g/ml l-ascorbic acid at 37 ◦ C, in 5% CO2 , and in 95% humidity as described ear¨ lier (Pasonen-Seppanen et al., 2001a,b). After 21 days, REKs produced morphologically well-organized epidermis with an effective barrier in the stratum corneum. The integrity of the barrier was monitored in permeation experiments with radiolabeled [3 H]-corticosterone and [3 H]-mannitol as described ¨ earlier (Pasonen-Seppanen et al., 2001a; Suhonen et al., 2003).

2.3.

Human epidermal membrane

Excised skin from the abdomen of a human cadaver was obtained from the Kuopio University Hospital (Kuopio, Finland) with the permission of the Board of Medicolegal Affairs. The epidermis was separated by heating the skin sample in distilled water at 60 ◦ C for 2 min. The epidermis was then peeled off and cut into 4.7 cm2 circular pieces. Separated skin samples were dried at room temperature under a gentle air flow for 1–2 days and stored at −20 ◦ C for later use.

2.4.

Formulations

Two combinations of permeation enhancers were investigated: sodium dodecyl sulfate (SDS):phenyl piperazine (PP) and methyl pyrrolidone (MP):dodecyl pyridinium chloride (DPC). These combinations were chosen because both are known to be potent enhancers but differ in terms of their safety; MP:DPC combinations are usually more irritating than SDS:PP (Mitragotri, S., personal communication). The enhancers were combined to form binary mixtures using four different total concentrations (0.5, 1, 1.5 and 2% (w/v)). At each total concentration the weight fractions of the enhancers varied between 0 and 1 in steps of 0.2. To ensure complete solubilization of each enhancer, the mixtures were prepared in 50% ethanol:PBS solution.

2.5.

Impedance measurement

The impedance measurements were conducted with a custom-made 7-hole Teflon chamber system, which was made to fit into the Transwell® culture plate well (Fig. 1). The cham-

Fig. 1 – 7-Hole (∅ 4 mm) Teflon chamber system for impedance measurement from the top (left), and the side profile of chamber in the 6-well culture plate under the buffer solution (right). A flat silver chloride electrode (∅ 3 cm) was placed underneath the chamber and another stick-shaped electrode was placed sequentially, from one hole to the next successively for the impedance measurements.

bers were constructed in the Technical Department of the University of Kuopio. The diameter of each hole was 4 mm. Fresh 3-week-old ROCs (4.7 cm2 ) or human skin samples of similar size (hydrated overnight in PBS) were placed between the chambers, and the parts were tightly clamped together with three screws. Human skin and ROC samples were pretreated in the Teflon chambers with enhancer formulations for 22 h. The stratum corneum part of the skin, 557 ␮l/cm2 of formulation was applied to the donor chamber, and the receiver chamber was filled with PBS buffer. The formulation and the PBS buffer were removed after 22 h, and the surface of the stratum corneum was gently washed with PBS to ensure complete removal of the formulation. Fresh PBS buffer was applied to donor and receiver chambers prior to measurement of electrical impedance at two time points: 0 and 22 h. To avoid contamination of the electrodes with enhancer components that might affect the signal, the measurement was always run in fresh PBS instead of in the presence of the enhancer formulations. A thin, flat silver chloride electrode with a diameter of about 3 cm was placed into the receiver chamber underneath the skin in such a way that it covered all seven holes of one chamber and was in contact with the PBS buffer (Fig.1). Another stick-shaped silver chloride electrode in the donor chamber was placed sequentially from one hole to another, and skin impedance readings were recorded with a Solatron 1286 four-electrode potentiostat and Solatron 1170 frequency response analyzer (Solatron Analytical, Hampshire, England). Sinusoidal voltage with an amplitude of 10 mV and frequency in the range of 0.05 Hz–60 kHz was applied to the system. The electrical impedance was presented and solved in the form of a Nyquist plot. The data points were fitted with an equivalent circuit model containing two resistors (R1, R2), a capacitor (C1) and a constant phase element (CPE), as described by Kontturi ¨ (1994) (Fig. 2). and Murtomaki The diameter of the semicircle in the Nyquist plot (Fig. 3) represents the skin membrane resistance (R1) of the equivalent circuit (Fig. 2) as a descriptor of skin permeability (Kontturi

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Fig. 2 – Equivalent circuit for skin containing resistors (R1, R2), capacitor (C1) and constant phase element (CPE).

¨ and Murtomaki, 1994). Enhancement of skin resistance was calculated as an impedance enhancement factor (EF) (Eq. (1)). EF =

R10 h R122 h

(1)

Overall, 48 permeation enhancers as binary mixtures of SDS:PP and MP:DPC were investigated.

2.6.

Drug permeation experiments

Forty-eight formulations were tested in impedance measurements, and 16 of these formulations were chosen for the subsequent drug permeation experiments. The permeation experiments were performed using side-by-side diffusion chambers. To ensure that there was a steady-state concentration of enhancer within the stratum corneum during the experiment, the epidermis was pre-equilibrated with the enhancer solution. In the same experiment, the permeation rates of a lipophilic corticosterone and a hydrophilic sucrose were investigated using double label detection of [3 H]corticosterone and [14 C]-sucrose. In a preliminary study, it was found that there was no mutual interference between these two labels in the analysis (data not shown). ROC inserts and briefly hydrated samples of human skin on 3 ␮m porous polycarbonate supporting membrane, similar to those used in the ROC culture inserts, were placed in between the side-by-side diffusion chambers thermostated at 37 ◦ C. Three milliliters of enhancer formulation were applied on both sides of the skin for 2 h. During that time, the solu-

Fig. 3 – Examples of a Nyquist plot of impedance (Z) on human skin and on ROC culture before (skin (), ROC (䊉)) and after (skin (), ROC ()) pretreatment with 50% EtOH in PBS.

243

tions in the donor and receiver chambers were changed every 30 min, which according to earlier experiments was sufficient to equilibrate the skin with the permeation enhancer. After 2 h of pretreatment, 3 ml of the enhancer formulation spiked with corticosterone (1 ␮l/ml) and sucrose (6 ␮l/ml) was applied to the donor chamber and 3 ml of fresh enhancer formulation was applied to the receiver chamber. Samples were taken from the receiver (200 ␮l) and donor (10 ␮l) compartments for 60 h; and to maintain a constant volume of the receiver, a similar volume of pure formulation was replaced. The samples were mixed with 3 ml of scintillation cocktail (UltimaGold, Packard, Bioscience, Groningen, The Netherlands) and analyzed with a liquid scintillation counter (WinSpectral 1414, Wallac, Finland). The permeability coefficient (P, cm/h) for a drug permeating through the skin or ROC model was approximated by a modification of Fick’s first law (Eq. (2)), P=

1 dQ AC dt

(2)

where A is the diffusional area (0.64 cm2 ), C the concentration of donor drug (dpm/ml), and dQ/dt is the rate of drug flux (dpm/h) that pass per unit area through the skin membrane. Permeability enhancement factor (EF) was calculated according to Eq. (3), EF =

Penhancer Pcontrol

(3)

where P is a drug permeability in enhancer and control (50% EtOH/PBS) experiments.

2.7.

Skin irritation testing

The irritation caused by the enhancer formulations was assessed using a method based on lactate dehydrogenase enzyme (LDH) leakage and morphological perturbation as described in detail elsewhere (Pappinen et al., 2005). Among the 16 formulations tested in the permeation experiments, five formulations were chosen for skin irritation testing in the ROC. Unfortunately, SDS and DPC interfered with the LDH assay in total concentrations of 0.6 and 0.8%, respectively, or higher; and only lower concentrations could be used. Briefly, ROCs were exposed to test chemicals for 30 min by applying 21.3 ␮l/cm2 of each formulation directly onto the stratum corneum of the ROC at room temperature. The formulation was then removed, the culture surface was rinsed twice with PBS, the medium in the receiver chamber was replaced with 1.5 ml of fresh DMEM2 (with 10% FBS, 4 mM L-glutamine, 50 U/ml penicillin and 50 g/ml streptomycin sulfate), and the plates were placed on a microplate shaker in an incubator (37 ◦ C, 5% CO2 , 95% humidity). After 8 h, a sample was withdrawn from the receiver chamber and stored at −70 ◦ C before analysis. For histology, the cultures were removed from the inserts, rinsed with PBS and fixed in Histochoice overnight at +4–8 ◦ C. The cultures were then embedded in paraffin and cut into 3 ␮m thick vertical sections prior to hematoxylin–eosin staining as described earlier (Pappinen et al., 2005). LDH release was determined using the commercial CytoTox 96® assay. The total available LDH enzyme was determined by lysing two control (untreated) cultures in each experiment.

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The results were calculated according to standard curves, and LDH release was compared to maximum LDH release (Eq. (4)). %LDH release =

sample LDH (U/l) × 100% maximum LDH (U/l)

(4)

Reproducibility of the LDH release assay compared to our earlier experiments was controlled by using two chemicals, aminotriazole and 1-bromopentene during the course of experiments. These compounds have evoked a mean LDH release of 0.6% (±0.1, S.E.M.) and 70.7% (±1.9, S.E.M.), respectively (Pappinen et al., 2005).

3.

Results

3.1.

Impedance measurements

Table 1 – Membrane resistance (R1), impedance enhancement factor (EF) and drug permeation in ROC and in human skin

Reports in the literature often report large standard deviations in impedance measurements, and that was also the case in our study (Fig. 4a). However, the electrical impedances of both human skin and ROC membrane were successfully fitted with the same equivalent circuit (Fig. 2). The fitting parameters were rather similar in both membranes (data not shown),

Skin Resistance before treatment (k cm−2 ) Resistance after treatment (k cm−2 ) Impedance EFa

a

95.28 ± 4.84

15.14 ± 2.85

30.36 ± 6.32

3.05 ± 0.43

3.17 ± 0.31

3.02 ± 0.55 0.31 ± 0.15

9.43 ± 0.87 4.75 ± 0.47

were

treated

with

50%

EtOH

in

PBS

EF = enhancement factor (R1 () 0 h/R1 () 22 h).

but a more shallow semicircle (Fig. 3), corresponding to a lower value of the frequency exponent ˛ (Kontturi et al., 1993), was observed for the ROC membrane. The diameter of the semicircle in the Nyquist plot (Fig. 3), which represents the average resistance (R1), of the ROC membrane was found to be slightly higher than that for human skin (Table 1). After the 22-h treatment with 50% EtOH:PBS, the control solution in this study, a 3-fold decrease in the resistance (R1) was seen in both membranes (Table 1). The profile of the impedance semicircle was found to remain similar after treatment with 50% EtOH:PBS (Fig. 3) and was not altered by any of the permeation enhancer binary mixtures (data not shown). In general, the permeation enhancers used in the present study were found to decrease the resistance (R1) of both membranes, and ROCs relative response to permeation enhancers was comparable to human skin (Fig. 4). A lesser correlation between enhancer treated human skin and ROC was observed both in the case of resistance (R2 = 0.623) and impedance EF (R2 = 0.588). There were marked permeation enhancer effects of the SDS:PP combinations, and similar, but not identical, effects on the human skin and ROC (Fig. 4). Almost all of the MP:DPC combinations decreased ROC resistance, but they did not alter human skin resistance as effectively, and only half of these combinations were more potent in human skin than the control.

3.2.

Fig. 4 – Correlation of impedance resistance (R2 = 0.623) (a) and enhancement factor (EF) (R2 = 0.588) (b) values between the ROC and human skin (, SDS:PP; 䊉, MP:DPC; , control) (mean ± S.E.M., n = 6–11).

55.99 ± 3.68

Permeability (10−4 cm/h) Corticosterone Sucrose Both membranes (mean ± S.E.M.).

ROC

Drug permeation experiments

The steady-state phase was reached and donor concentrations were maintained throughout 20–60 h, confirming the integrity of the membranes during the experiments (Fig. 5). For both model solutes in 50% EtOH:PBS, the ROC membrane was found to be more permeable than human skin (Table 1). Corticosterone penetrated three times faster and for sucrose there was a 15-fold difference in favour of ROC compared to human skin. The enhancer formulations increased the permeability coefficient for corticosterone by 40-fold in human skin and by 12-fold in ROC (Fig. 6a). In human skin, sucrose permeation was accelerated as much as 200-fold, while through ROC the increase, thought still significant, was only 18-fold (Fig. 6b). Almost all 16 formulations used in the permeation studies were found to increase the permeability of both membranes

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245

not linear (Fig. 6d). ROC was found to be more sensitive to the less potent permeation enhancers than human skin, this being especially evident for the hydrophilic sucrose.

3.3. Correlation of skin impedance with drug permeation

Fig. 5 – Cumulative sucrose permeation profiles through the human skin (䊉) and ROC culture () during treatment with the most effective permeation enhancer, 2% SDS:PP 0.4:0.6.

(Fig. 6). The difference in the permeability decreased in the presence of permeation enhancers, particularly in the case of more efficient enhancers (Table 2). A linear correlation (R2 = 0.953) between the permeabilities of ROC and isolated human skin was obtained with corticosterone (Fig. 6c). Although the correlation coefficient for sucrose permeation in ROC and human skin was 0.830, the relationship was curved,

Steady-state drug permeation was attained at about 20 h, and therefore 22 h was chosen as an endpoint for the impedance measurement. In general, there was some correlation between drug permeability and electrical impedance in both membranes. However, in the ROC model, the resistance (R1) was found to correlate well with permeability (corticosterone, R2 = 0.811; sucrose, R2 = 0.714) while in human skin there was no clear linear relationship (corticosterone, R2 = 481; sucrose, R2 = 0.435) (Fig. 7a and b). One formulation (1.5% PP) was clearly an outlier in the ROC data, having a marked effect on drug permeation, but only a marginal effect on electrical impedance. If this formulation were to be exluced, then the correlation coefficients for corticosterone and sucrose permeation with resistance (R1) in ROC would be R2 = 0.939 (Fig. 7a) and R2 = 0.842 (Fig. 7b), respectively; and the correlation with EF values would be R2 = 0.938 (Fig. 7c) and R2 = 0.893 (Fig. 7d), respectively. According to introduced porous pathway theory, the permeability (P) of hydrophilic and ionic solutes across the skin

Fig. 6 – Permeability enhancement ratio (EF) (Penhancer /Pcontrol ) (a, b) and permeabilities (c, d) in ROC and the human skin; corticosterone (a, c) and sucrose (b, d). Formulations with different concentrations (w/v) and volume fractions; SDS:PP 2% 0.4:0.6 (), SDS:PP 1.5% 0:1 (×), SDS:PP 2% 0.6:0.4 (—), SDS:PP 1.5% 0.4:0.6 (䊉), SDS:PP 1% 0.4:0.6 (), SDS:PP 0.5% 0.2:0.8 (), SDS:PP 1% 0.8:0.2 (*), MP:DPC 2% 0:1 (), SDS:PP 0.5% 0.6:0.4 (+), MP:DPC 1% 0:1 (◦), MP:DPC 0.5% 0.2:0.8 (−), MP:DPC 2% 0.6:0.4 (), MP:DPC 1% 0.6:0.4 (♦), MP:DPC 0.5% 0.6:0.4 (), MP:DPC 0.5% 0.8:0.2 (), MP:DPC 1% 1:0 (), 50% EtOH:PBS (•) (ka, n = 6–9).

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Table 2 – Ratio of drug permeability (P) in ROC to isolated human skin, and the rank order of formulation effect according to permeation data Formulation

Sucrose

Corticosterone

PROC /PSkin

EffectSkin

EffectROC

PROC /PSkin

EffectSkin

EffectROC

SDS:PP 2.0% (0.4:0.6) 1.5% (0:1) 2.0% (0.6:0.4) 1.5% (0.4:0.6) 1.0% (0.4:0.6) 0.5% (0.2:0.8) 1.0% (0.8:0.2) 0.5% (0.6:0.4)

1.40 1.73 2.60 4.17 15.24 18.68 8.24 5.00

1 2 3 4 7 8 6 5

1 3 2 4 5 7 6 8

1.06 0.99 1.15 1.40 1.62 2.04 2.65 2.74

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

50% EtOH:PBS

15.44

9

9

3.13

9

9

MP:DPC 2.0% (0:1) 1.0% (0:1) 1.5% (0.2:0.8) 2.0% (0.6:0.4) 1.0% (0.6:0.4) 0.5% (0.6:0.4) 0.5% (0.8:0.2) 1.0% (1:0)

33.04 4.13 29.46 19.90 2.30 46.27 17.08 12.59

4 2 6 5 1 9 7 3

1 2 3 4 6 7 8 5

1.80 2.10 2.31 1.53 2.74 2.80 1.96 2.26

1 2 4 3 6 7 5 8

1 2 3 4 5 6 7 9

50% EtOH:PBS

15.44

8

9

3.13

9

8

can be related to skin impedance (R) according to Eq. (5), log P = log C − log R

(5)

where C is a constant that depends on the solute radius and the structure of stratum corneum. The theory is described in more detail in the literatures (Li et al., 1998, 1999; Tang et al., 2001; Tezel et al., 2002, 2003). For transport via a porous pathway, log P versus log R relationship should be linear with a slope of −1. In this study, the correlation of sucrose permeability with skin impedance in human skin did have a slope of −1.00 (Fig. 7b), whereas in the ROC the slope was −0.61 (Fig. 7a). The slopes of lipophilic corticosterone, as expected, deviated from −1 being −0.69 in human skin and −0.60 in ROC (Fig. 7b and a).

3.4.

Irritation of enhancer formulations

The extents of LDH release evoked by the control chemicals aminotriazole (non-irritant) and 1-bromopentene (irritant) were 0.6 ± 0.3 and 62.3 ± 3.0%, respectively, which was comparable to earlier data (Pappinen et al., 2005) and confirmed the validity of ROC in this study. The 50% EtOH:PBS caused LDH leakage of only 3.1 ± 1.5%. We have previously defined chemicals as being “slightly irritant” if LDH release was more than 25% and “irritant” if LDH release was over 50% (Pappinen et al., 2005). According to this classification, only 1% MP:DPC 0.6:0.4 would be predicted as being slightly irritating (Fig. 8). The LDH release data indicates that all of the other enhancer combinations should be classified as non-irritant. This irritation seems to be attributable to the presence of DPC because 1% MP alone did not cause any significant LDH release (1.9%).

Aminotriazole, 1% MP:DPC 1:0, 1% SDS:PP 0.4:0.6 and 50% EtOH:PBS treated cultures did not evoke morphological changes on the culture structure, though we did observe some condensed cell nuclei in basal and spinous cell layer (Fig. 9). In contrast, 1% MP:DPC 0.6:0.4 and 1-bromopentene treatments caused severe epidermal degeneration with cell death and blebbing to the collagen gel. The morphological perturbations seen with 0.5% MP:DPC 0.8:0.2 and 1.5% SDS:PP 0:1 were between these two extremes, having rather normal morphology with some apoptotic cells, cell vacuolization and condensed nuclei. Overall, the rank order of morphological perturbation and LDH release were identical.

4.

Discussion

The resistance of the human epidermal membrane (HEM) has been observed to lie in the range of 1–120 k cm−2 (DeNuzzio and Berner, 1990; Sims et al., 1991; Kontturi et al., 1993; Inada et ¨ 1994). Various experimental al., 1994; Kontturi and Murtomaki, conditions, such as the hydration level of the skin (Kalia and Guy, 1995), the ionic strength of the bathing medium (Curdy et al., 2002), the frequency and current density (Yamamoto and Yamamoto, 1977, 1978; Rosell et al., 1988), the skin surface area (Fasano et al., 2002), the impedance measurement equipment and the operational settings (Fasano and Hinderliter, 2004), are likely to affect the resistance of skin, and lead to the substantial variation reported in the data. The high intra- and interindividual variation in skin electrical impedance is a very typical feature of normal skin (Yamamoto and Yamamoto, 1977; Rosell et al., 1988) and also after chemical treatment (Jain et al., 2006; Karande et al., 2006). Large standard devi-

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Fig. 7 – Correlation of impedance resistance with corticosterone (䊉) and sucrose () permeabilities in the ROC (a) and in the human skin (b), and correlation of impedance EF value with permeability of corticosterone (c) and sucrose (d) in the ROC. Formulations with different concentrations (w/v) and volume fractions (c, d); SDS:PP 2% 0.4:0.6 (), SDS:PP 1.5% 0:1 (×), SDS:PP 2% 0.6:0.4 (—), SDS:PP 1.5% 0.4:0.6 (䊉), SDS:PP 1% 0.4:0.6 (), SDS:PP 0.5% 0.2:0.8 (), SDS:PP 1% 0.8:0.2 (*), MP:DPC 2% 0:1 (), SDS:PP 0.5% 0.6:0.4 (+), MP:DPC 1% 0:1 (◦), MP:DPC 0.5% 0.2:0.8 (−), MP:DPC 2% 0.6:0.4 (), MP:DPC 1% 0.6:0.4 (♦), MP:DPC 0.5% 0.6:0.4 (), MP:DPC 0.5% 0.8:0.2 (), MP:DPC 1% 1:0 (), 50% EtOH:PBS (•) (mean, n = 6–11).

ations in the impedance measurements were observed also in our study. Skin exhibits non-ohmic behaviour, meaning that skin resistance decreases with increasing current or voltage (Kasting and Bowman, 1990). The electrical characteristics of

Fig. 8 – LDH leakage after 8 h exposure to enhancer formulations, aminotriazole, 1-bromopentene, and 50% EtOH:PSB solution (mean ± S.E.M., n = 6–9).

the skin can be presented as equivalent circuit models, usually composed of resistors and capacitors (Yamamoto and Yamamoto, 1977, 1978; van Boxtel, 1977; Kalia and Guy, 1995, 1997). The refined equivalent circuit model of Kontturi and ¨ (1994) described our data best. In that model, one Murtomaki capacitor element is replaced by a constant phase element (CPE) (Fig. 2). The values of human skin resistance (55 k cm−2 ) observed in the present study were similar to those previously ¨ reported (Kontturi et al., 1993; Kontturi and Murtomaki, 1994), and the resistances of ROC model (95 k cm−2 ) and human skin (55 k cm−2 ) were also in agreement with the values in the literature (DeNuzzio and Berner, 1990; Sims et al., 1991; Inada et al., 1994). Although the skin resistance in the ROC model was higher than in the human skin, both membranes exhibited a similar depressed semicircle shape in the Nyquist plot (Fig. 3). Chemical permeation enhancers usually interfere with the organized lipid bilayer structures in the intercellular spaces between the corneocytes, leading to disruption in the integrity of these structures and thereby facilitating the permeation of solutes through the skin (Suhonen et al., 1999). In this way, permeation enhancers alter the skin electrical resistance ¨ (Kontturi and Murtomaki, 1994; Kalia and Guy, 1997) and this was also seen in our data. Based on the Nyquist plots, both

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Fig. 9 – The morphology of penetration enhancer treated ROC cultures. After exposure to the test chemicals, ROC cultures were fixed, processed for histology and stained with hematoxylin and eosin. Control cultures (a) showed well-organized epidermal structure with thick stratum corneum. The morphology of aminotriazole (b), 1% MP:DPC 1:0 (c) and 1% SDS:PP 0.4:0.6 (e) treated cultures was rather similar to that seen in control cultures except that there were some condensed cell nuclei in the basal and spinous cell layer (arrows). One percentage MP:DPC 0.6:0.4 (d) and 1-bromopentene (f) treatments caused severe epidermal degeneration (arrowheads). Scale bar 50 ␮m.

ROC and human skin reacted qualitatively in a similar way to the permeation enhancers. The permeability coefficients were in the same range in the human skin (corticosterone 0.4–10.9 (10−3 cm/h), sucrose 0.02–6.0 (10−3 cm/h)) and in the ROC (corticosterone 0.9–11.5 (10−3 cm/h), sucrose 0.5–8.4 (10−3 cm/h)) after permeation enhancer treatment. However, a linear correlation between the two membranes was observed only for corticosterone (Fig. 6c). The ROC model was found to predict reasonably the rank order of corticosterone permeation from the formulations (Table 2). The lipophilic drug corticosterone (Log Koctanol/water 1.94) is believed to permeate through the lipid matrix of the skin. Unlike corticosterone, sucrose is very hydrophilic (Log Koctanol/water −3.7) and thus it permeates poorly through the skin. In human skin, only the most effective enhancers were able to significantly increase sucrose permeation, but in the ROC most formulations increased sucrose permeation. This observation may be related to the less organized lipid structure of the intercellular space of stratum corneum in ROC (unpublished data), which may be more readily perturbed with the creation of new polar permeation routes through the stratum corneum. Lipophilic solutes permeate through the stratum corneum via intercellular lipid domains whereas hydrophilic solutes favour the aqueous pores in the stratum corneum. Accord-

ing to porous pathway theory (Li et al., 1998, 1999) the plot of hydrophilic solute permeability against skin impedance should have a slope of −1, which suggest similar transport pathways for the hydrophilic permeant and conducting ions across the skin (Eq. (4)). In human skin sucrose permeability was related with skin impedance with the slope value of −1.00 supporting the porous pathway theory in the presence of permeation enhancers. However, significant deviation from a slope of −1 was observed in ROC (−0.61) which is interpreted to be due to the presence of additional pathways. As discussed in the previous paragraph, the lipid organization of ROC is more disordered (unpublished data) and seems to be more susceptible to permeation enhancers than human skin. Therefore hydrophilic solutes cannot be exclusively penetrating through aqueous pores, but are also able to diffuse through the lipid domain. However, it is not clear whether the lipophilic solutes utilize the same diffusion pathway as hydrophilic molecules or ions in the presence of permeation enhancers. According to our data, corticosterone did not use solely the porous pathway theory (slope values of 0.69 and 0.60 for human skin and ROC, respectively), but permeability correlated with electrical impedance. Karande et al. (2006) obtained different results. However, Karande et al. (2006) used a finite dose applied to the donor chamber instead of a symmetric enhancer configuration. The permeation curves resulting in

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this setup are generally non-linear and the analysis of permeation rates will be more difficult. We have also seen that the finite dose system results in different data than the symmetrical setup (data not shown). Although finite dose application is a more clinical approach, to undertake an appropriate mechanistic analysis of the drug permeation data, it is necessary that the skin is equilibrated with enhancers to avoid transient enhancer concentration gradients within the skin. In this study, we have demonstrated for the first time the correlation between drug permeability and electrical impedance in human skin, but the correlation was not linear. In ROC, drug permeation correlated very well with the impedance data, except for one outlier (1.5% phenyl piperazine that markedly increased drug permeation without having any significant effect on electrical impedance). The fraction of appendages, such as sweat glands and hair follicles, in human skin is low (0.1%) and as electrical impedance is measured on a very small surface area (0.125 cm2 ), the number of appendage routes may become important leading to the low correlation and high variation in permeability, which is characteristic of human skin. ROC is a non-porous membrane without the appendages, and this might be the reason for the better correlation with this model system. Permeation enhancers are often skin irritants, and the safety of topical formulations must be tested before use. Nowadays chemical safety on skin is still evaluated in vivo using the rabbit skin irritation test (Draize et al., 1944), but alternative methods such as cultured skin models are being developed (Fentem et al., 2001; Portes et al., 2002; Heylings et al., 2003). Our recent report shows that ROC can be used as an in vitro skin irritation model for chemicals by using irritation markers, such as lactate dehydrogenase leakage (LDH), interleukin ␣ release and morphological perturbation (Pappinen et al., 2005). In the present study, the cell membrane integrity (LDH assay) and the epidermal tissue morphology were evaluated, and the irritation attributable to five different formulations was defined. Only one of these formulations was predicted to be a slightly irritating (1% MP:DPC) based on LDH release, and it was also found to cause severe morphological degradation. The other formulations were classified as non-irritants according to LDH release, and most of them caused mild to moderate disturbances in epidermal morphology. The rank order of irritation was found to be similar whether assessed by LDH release or morphological perturbation. In conclusion, the effects of permeation enhancers were found to be similar in the human skin and in the ROC model, but a linear correlation of skin electrical impedance with drug permeability was seen only in ROC, not in human skin. ROC seems to react more consistently to permeation enhancer formulations and thus, may be applicable as a viable surrogate for human skin in pharmaceutical studies. For the first time we demonstrated that the enhancer effect and enhancer induced irritation can be tested using an artificial skin model.

Acknowledgements The present study was supported by the Graduate School of Electrochemical Science and Technology of Polymers and

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Membranes including Biomembranes (ESPOM). The authors wish to thank Dr. Samir Mitragotri for his help in choosing the enhancer mixtures for this study.

references

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