Effects of Oxygen-Containing Terpenes as Skin Permeation Enhancers on the Lipoidal Pathways of Human Epidermal Membrane

Effects of Oxygen-Containing Terpenes as Skin Permeation Enhancers on the Lipoidal Pathways of Human Epidermal Membrane DOUNGDAW CHANTASART,1 THANED PONGJANYAKUL,2 WILLIAM I. HIGUCHI,3 S. KEVIN LI4 1

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand

2

Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

3

Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112

4

Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati, Cincinnati, Ohio 45267

Received 28 May 2008; revised 23 September 2008; accepted 16 November 2008 Published online 20 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21666

ABSTRACT: The present study investigated the effects of oxygen-containing terpenes as skin permeation enhancers on the lipoidal pathways of human epidermal membrane (HEM). The enhancement (EHEM) effects of menthol, thymol, carvacrol, menthone, and cineole on the transport of a probe permeant, corticosterone, across HEM were determined. It was found that the enhancer potencies of menthol, thymol, carvacrol, and menthone were essentially the same and higher than that of cineole based on their aqueous concentration in the diffusion cell chamber at EHEM ¼ 4. Thymol and carvacrol also had the same EHEM ¼ 10 concentration further supporting that they had the same enhancer potency based on the aqueous concentration. The uptake amounts of terpene into the HEM stratum corneum (SC) intercellular lipid under the same conditions indicate that the intrinsic potencies of the studied terpenes are the same based on their concentration in the SC and similar to those of n-alkanol and n-alkylphenyl alcohol. Moreover, they are all better enhancers compared to branched-chain alkanol. The approximately same uptake enhancement of b-estradiol induced by the studied terpenes and alcohols at EHEM conditions into the SC intercellular lipids suggests that the mechanism of enhancement action for the terpenes and those of alcohols are essentially the same. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3617–3632, 2009

Keywords: oxygen-containing terpenes; skin permeation enhancers; human epidermal membrane; permeant; lipoidal pathway

INTRODUCTION It is generally accepted that the principal barrier to most transdermal drug delivery is the stratum corneum (SC), the outermost layer of the skin

Correspondence to: Doungdaw Chantasart (Telephone: þ66 26448677 ext. 1309; Fax: þ66 26448694; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 3617–3632 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association

comprising keratin-rich cells embedded in multiple lipid bilayers. Overcoming the barrier by using skin permeation enhancers has been one of the great interests in pharmaceutical research. Skin permeation enhancers are defined as chemicals which are themselves pharmacologically inactive, but can partition into and interact with the barrier of the SC when incorporated into a transdermal formulation, thereby reducing the resistance of skin to drug transport.1–4 An ideal skin permeation enhancer should be nonirritating

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3617

3618

CHANTASART ET AL.

and should not alter the skin irreversibly. Many chemical enhancers such as dimethyl sulfoxide, surfactants, alcohols, and urea and its derivatives have been studied for their permeation enhancement.4–8 The adverse effects caused by some of these enhancers restrict their wide usage. Currently, there has been an upsurge in the use of naturally occurring chemicals such as terpenes as skin permeation enhancers. Terpenes are a safe and effective class of skin permeation enhancers derived from plant essential oils. The US Food and Drug Administration classified them as ‘‘General Regarded As Safe’’ (GRAS).9 They were reported to have good toxicological profiles, high percutaneous enhancement abilities, and low cutaneous irritancy at low concentrations (1–5%).10,11 Terpenes are made up of various chemical structural classes such as hydrocarbons, alcohols, ketones, and oxides.12 A variety of terpenes have been reported to increase the percutaneous absorption of both hydrophilic13–15 and lipophilic drugs.16–18 For example, menthol, cineole, menthone, geraniol, carvacrol, and limonene have been used to enhance the permeation of drugs including 5-fluorouracil,15 propranolol hydrochloride,13 zidovudine,14 diclofenac sodium,16 azidothymidine,17 and indomethacin.18 Although a number of studies have investigated the penetration enhancement effects of terpenes, the underlying mechanisms of action of terpenes as skin permeation enhancers are not clearly defined. In the past two decades, a research focus in the Higuchi’s group has been to understand the mechanisms of action of skin chemical permeation enhancers.19–24 Corticosterone (CS) was selected as the model permeant as it has been shown to be particularly suitable for quantitatively probing the lipoidal pathway of the hairless mouse skin (HMS) SC using a parallel pore and lipoidal skin transport pathway model.19–21,25 b-Estradiol (E2b), a highly lipophilic steroidal drug (log Koctanol/water ¼ 4.01), was selected as the surrogate permeant for CS in equilibrium uptake studies due to the convenience and the sensitivity of E2b in SC uptake measurement; the quantification of CS (log Koctanol/water ¼ 1.94) in the SC intercellular lipids was difficult. The use of E2b and CS interchangeably in the permeation and uptake studies was supported by previous studies.25,26 Isoenhancement concentrations were defined as the aqueous concentrations for which different enhancers induce the same extent of transport enhancement of the steroidal model permeant

across the SC lipoidal pathway. For example, the isoenhancement concentration of E ¼ 10 is the aqueous concentration at which an enhancer can induce a tenfold flux enhancement (E) for a permeant. In these previous studies, the effects of the enhancers upon the transport of CS across the lipoidal pathway of HMS were determined and the equilibrium uptake of the enhancers into the SC intercellular lipid domain was measured. The results showed a general quantitative structure– enhancement relationship for the chemical permeation enhancers. It was suggested that the microenvironment of the enhancer site of action in HMS can be well-mimicked by liquid n-octanol. These previous studies also showed that the potency of an enhancer is related to its lipophilicity based on the enhancer aqueous concentration, but the intrinsic potency of the enhancer is relatively independent of its lipophilicity when compared using the equilibrium concentrations of the enhancers in the SC intercellular lipids. Later, Chantasart et al.,27 used the same transport and uptake experimental methods with human epidermal membrane (HEM) to test the hypothesis that HMS can be a reliable model for the evaluation of the effects of the skin permeation enhancers on the lipoidal pathway of HEM. The HEM studies support the hypotheses made in the previous HMS studies. The objectives of the present study were to (a) assess the effects of oxygen-containing terpenes on permeant transport across the SC lipoidal pathway in HEM, (b) determine the environment of the site of enhancer action of terpenes in HEM and compare it with that in our previous studies,19,23,27 (c) examine whether the hypotheses made in these previous studies would continue to hold with a new class of chemical enhancers, the oxygen-containing terpenes, and (d) provide mechanistic insights of terpenes as skin permeation enhancers. Oxygen-containing terpenes were chosen for the present study because they are naturally occurring volatile oils that appear to be promising candidates for use as clinically acceptable enhancers.15 Menthol, thymol, carvacrol, menthone, and cineole are alcohol, ketone, and oxide terpenes that were to be studied for their skin permeation enhancing effects, and their structures are shown in Figure 1. The present study employed the same experimental design as that in a previous HEM study.27 CS was selected as the probe permeant for the transport experiments and E2b was to be used for the equilibrium uptake studies. The permeability

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

3619

PO4  2H2O, 7.57 g Na2HPO4, 4.40 g NaCl, and 0.2 g NaN3 in 1 L distilled water.28 Terpene solutions (terpene/PBS solutions) at different terpene concentrations were prepared by dissolving the terpenes in PBS. The terpene concentrations used in the present study were lower than the aqueous solubilities of the terpenes.

Preparation of HEM

Figure 1. The structures of the studied terpenes.

coefficients of the lipoidal pathways of HEM SC for CS at different aqueous terpene concentrations were to be determined. The isoenhancement concentrations of the terpenes at E ¼ 4 and E ¼ 10 for HEM (EHEM ¼ 4 and EHEM ¼ 10, respectively) were calculated, and the amounts of the terpenes in the intercellular lipids of human SC were determined at the isoenhancement concentrations.

Human skin was obtained from abdominoplastic surgical operations (Department of Surgery, Yanhee General Hospital, Thailand) of female patients aged between 35 and 75 years within a few hours after operation. HEM, which includes the SC and viable epidermis, was separated from the dermis by heat separation technique.29 Briefly, the skin samples freed from fatty tissue were immersed in water at 608C for 60 s. After heat treatment, the epidermis sheet was separated from the dermis by roll-peeling using plastic forceps.30 The HEM was soaked in PBS, blotted dry, wrapped in aluminum foil, and stored at 208C for later use. The described experimental protocol was approved by the committee on human rights related to human experimentation, Mahidol University, Bangkok, Thailand.

HEM Permeability Experiments

EXPERIMENTAL METHODS Materials Corticosterone (CS), thymol, menthol, 1-octanol, 4-octanol, 2-phenylethanol, and sodium azide (NaN3) were purchased from Fluka Chemika (Milwaukee, Switzerland). Menthone was purchased from Alfa Aesar (Ward Hill, MA). Carvacrol, cineole, b-estradiol (E2b), and trypsin from bovine pancreas were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium dihydrogen orthophosphate dihydrate (NaH2PO42H2O) and disodium hydrogenphosphate anhydrous (Na2HPO4) were purchased from Ajax Finechem (NSW, Australia). Absolute ethanol was purchased from Carlo Erba Reagent (Val de Reuil, France). Sodium chloride (NaCl), high pressure liquid chromatography (HPLC) grade methanol (MeOH), chloroform (CHCl3) and n-heptane were purchased from Labscan Asia Co., Ltd (Bangkok, Thailand). Phosphate buffered saline solution (PBS) pH 7.4 containing 0.02% NaN3 was prepared by dissolving 2.10 g NaH2 DOI 10.1002/jps

The permeability experiments were carried out in a two-chamber side-by-side diffusion cell with HEM. Prior to mounting the HEM samples in the diffusion cells, the frozen skin samples were allowed to thaw at room temperature and hydrate overnight in PBS. Each HEM was mounted between the two diffusion half-cells with a regenerated cellulose membrane (Spectra/Por1 MWCO 12,000–14,000, Spectrum Laboratories, Inc., Rancho Dominguez, CA) as a support placed between the viable epidermis side of the HEM sample and the receiver chamber. The cellulose support membrane was previously soaked in PBS overnight and had permeability coefficient several orders of magnitude higher than that of HEM. This cellulose membrane could minimize possible damage resulting from physical stress placed upon the HEM during the experiment. Each diffusion cell compartment had a 2-mL volume and an effective diffusional area of around 0.71 cm2. Two milliliters of PBS were pipetted into both chambers. The diffusion cell was placed in a circulating water bath at 37  18C for 12 h. In the terpene experiments, to

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3620

CHANTASART ET AL.

achieve equilibrium of the terpene with HEM, the terpene/PBS solution in both chambers was replaced several times (nine times of 20 min each). Following terpene equilibration, saturated CS in the terpene/PBS solution was added to the donor chamber. The concentration of the terpene was checked before and at the end of each transport run to ensure that no significant depletion of terpene had taken place. Samples were withdrawn from the donor and receiver chambers at predetermined time intervals (e.g., 5, 7, 8, and 9 h). Typically, 10-mL aliquots were taken from the donor chamber and 500-mL aliquots were withdrawn from the receiver chamber. The same volume of the fresh solution (same composition as the starting solution) was added back to the receiver chamber after each aliquot removal to maintain a constant volume. The samples were then diluted in mobile phase and were analyzed for CS by HPLC. Experiments were run long enough so that the duration of the experiments was around three to five times longer than the transport lag times. The total duration of the skin penetration experiment from the assembly of HEM in the diffusion cells to the completion of the experiment was around 24 h. The total permeability coefficient ( PT) was determined from the slope of a cumulative amount transport across the membrane versus time plot in the steady state region. Experiments conducted without the terpenes but with only PBS solution were the baseline control. CS metabolism in HEM during transport across the membrane under the present experimental conditions was expected to be minimal.23

HEM Electrical Resistance Measurements The electrical resistance of HEM was measured immediately before and after each transport experiment by applying a small voltage (<0.25 V) as previously described.27 An electrical system was built to measure the electrical resistance of HEM in the side-by-side diffusion cells. In this system, a 1.5-V battery was connected with a high resistance fixed resistor in series with the HEM using Ag/AgCl as the electrodes. The voltage across the HEM and the fixed resistor was determined by a voltmeter, and the resulting electric current was calculated by the voltage across the fixed resistor and the resistance of the resistor using Ohm’s law. The electrical resistance of HEM was then calculated using the voltage reading across the HEM and the electric current according to Ohm’s law. As the preparation of skin

membrane is quite laborious, the electrical resistance of HEM before each transport experiment served as the method to check for skin integrity. Previous studies27,31,32 suggest that HEM with electrical resistance >15–20 kV cm2 can be indicative of good integrity. Therefore, only HEM samples with initial resistance >15 kV cm2 were used in the present study.

Test for HEM Recovery At the end of each transport experiment at EHEM ¼ 4 concentrations for the studied terpenes and EHEM ¼ 10 concentrations for thymol and carvacrol, the solutions in the receiver and donor chambers were removed by pipette and rinsed with PBS for 3  20 min followed by 1  10 h overnight. Following the PBS rinsing, transport studies with PBS in both chambers were carried out using CS as the permeant. The permeability coefficients obtained in the PBS transport experiments after terpene-enhanced transport experiments at EHEM ¼ 4 and EHEM ¼ 10 concentrations of each terpene were then compared with those obtained with normal PBS.

Solubility Experiments Solubility of CS was determined as previously described20,33 by adding 2 mg of CS in 1 mL of an enhancer solution in Pyrex culture tubes (diameter, 13 mm; length 100 mm). The drug suspension was equilibrated for 72 h at 37  18C, shaken in a thermostatically controlled water-bath. After equilibration, the culture tubes were centrifuged at 3500 rpm for 15 min (Hettich Universal 30F, Tuttlingen, Germany) and the clear supernatants were analyzed for CS concentrations with HPLC. The solubility of E2b in all enhancer solutions was also determined in a similar manner at 1 mg of E2b in 2 mL of enhancer solution in Pyrex culture tubes.

Calculation of the Enhancement Factors The enhancement factor for transport across the lipoidal pathways of HEM (EHEM) was determined by:22,34    PL;X SX EHEM ¼ (1) PL;0 S0

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

where PL,X and PL,0 are the CS permeability coefficients for the lipoidal pathway when the solvent in both chambers is terpene/PBS and PBS, respectively. SX and S0 are the CS solubilities in terpene/PBS and PBS, respectively. For CS as a permeant and in the range of EHEM  10, PL;X  PT;X and PL;0  PT;0

(2)

where PT,X and PT,0 are the total permeability coefficients of HEM when the solvent is terpene/ PBS and when the solvent is PBS, respectively.20,22,33 PT,X and PT,0 were determined by:    1 dQ PT;X or PT;0 ¼ (3) ACD dt where A is the effective diffusion area of the diffusion cell, CD the concentration of the permeant in the donor chamber, and dQ/dt the slope of the steady-state region of the plot of cumulative amount of permeant transported into the receiver chamber versus time under sink conditions. Following the same experimental strategy used in the previous studies,22,25,33 the EHEM ¼ 4 and EHEM ¼ 10 isoenhancement concentrations were used to quantify the potency of the terpenes as permeation enhancers. The EHEM ¼ 4 and EHEM ¼ 10 isoenhancement concentrations of an enhancer are defined as the aqueous enhancer concentration in equilibrium with the SC in both donor and receiver chambers of the two-chamber diffusion cell that gives an enhancement factor of 4 and 10, respectively (Eq. 1). The EHEM ¼ 4 and EHEM ¼ 10 isoenhancement concentrations for each terpene were determined by plotting the enhancement factors against the terpene concentration and the interpolation of the results. The EHEM ¼ 10 isoenhancement concentrations were only determined for thymol and carvacrol due to the aqueous solubilities of the other terpenes.

Isolation of Human Stratum Corneum The SC was isolated using a previously published epidermal separation technique35 with some modifications. Briefly, HEM was placed viable epidermis side down on a filter paper (quantitative filter paper No.1, Whatman1) in a Petri dish. The Petri dish was covered with 0.0005% trypsin in PBS solution up to the surface of SC. The Petri dish was then covered and maintained at 37  18C for 18 h. After the trypsin treatment, the viable epidermis side of the SC was gently swabbed with a cotton bud. The SC was rinsed thoroughly with DOI 10.1002/jps

3621

distilled water several times and placed on a stainless steel mesh spatula. The SC was blotted with tissue paper held by tweezers to remove excess water. Then the SC was placed on aluminum foil and dried at room temperature. Before the SC was completely dry, it was separated from aluminum foil with tweezers so that it would not adhere to the aluminum foil. After complete drying at room temperature in a desiccator containing silica gel, the SC was kept in a freezer for later used.

Partitioning Study with Human SC Preparation of n-Heptane-Treated Human SC n-Heptane-treated SC was prepared as described in a previous study.27 Briefly, the SC (about 3– 4 mg) was carefully weighed and then rinsed in nheptane solvent for 3  10 s. Excess n-heptane on the SC surface was completely removed with tissue paper. The SC was put in a glass vial and dried in a desiccator containing silica gel at room temperature overnight. After complete drying, the n-heptane-treated SC was carefully weighed and kept in a tight glass vial for later use. The weight percentage of the surface lipid of SC was determined from the weight change of the SC after n-heptane treatment. Partitioning Experiments with n-Heptane-Treated Human SC Partition experiments were carried out to determine the uptake amounts of the studied terpenes and E2b partitioning into n-heptane-treated human SC using the method described in the previous study.27 SC (about 3–4 mg) was carefully weighed and soaked in about 20 mL of E2b saturated terpene solution at EHEM ¼ 4 and EHEM ¼ 10 concentrations in a screw capped glass vial. The vial was sealed with parafilm to prevent terpene evaporation and put in a thermostated water-bath with shaking at 37  18C for 12 h. After 12 h, the human SC was taken out from the solution by tweezers and blotted by tissue paper. Samples were taken from the screw-capped glass bottle to check for possible E2b depletion in the solution. The wet human SC was carefully weighed in a 10 mL snap-capped glass bottle. Three milliliters of 100% ethanol were added into the bottle to extract the enhancer and E2b from the SC for 48 h at room temperature with occasional gentle agitation. The extracted solution

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3622

CHANTASART ET AL.

and the solution in the screw-capped glass bottle were then transferred to a screw-capped Pyrex test tube. The test tube was centrifuged at 3500 rpm for 15 min (Hettich Universal 30F). The supernatant was analyzed for the terpene by GC. The uptake amount of terpene per unit dry weight of n-heptane-treated SC, Acorrected,i, was calculated as follows: Acorrected;i ¼

Aextracted;i Ci  ðWwet  Wdry Þ Wdry Wdry

(4)

where Aextracted,i is the amount of terpene extracted from n-heptane-treated HMS SC, Wdry is the dried n-heptane-treated SC weight, and the subscript i represents terpene. A correction for the terpene in the aqueous compartment(s) of SC was calculated according to the wet weight of SC (Wwet) and the concentration of the terpene in the aqueous bulk phase (Ci). This correction assumes that the hydration portion of the ‘‘wet’’ SC can be represented by the equilibrium bulk aqueous phase solution. The (n-heptane-treated SC/ aqueous phase) partition coefficient of E2b (KE2b) was calculated as follows: KE2b ¼

½A0extracted  ðWwet  Wdry ÞC0i =Wdry S0X (5) C0i S0O

temperature. The residue of SC was taken and rinsed several times with fresh CHCl3/MeOH (2:1) mixture and dried under room temperature for 24 h. The dried delipidized human SC after extraction was carefully weighed and kept in a desiccator containing silica gel. The intercellular lipid content was determined by the change in weight of the n-heptane-treated SC after solvent extraction. Enhancer partition experiments with delipidized human SC were carried out using the experimental procedure similar to that described in the ‘‘Partitioning Experiments with n-Heptane-Treated Human SC’’ Section. GC Analysis The GC system (Perkin Elmer, Norwalk, CT) consisted of injector, controller, and flame ionization detector (FID) connected to a 0.32-mm column ID, 0.25-mm film thickness, 30-m length, fused silica capillary column (Supelco, Bellefonte, PA), and temperature programming of 60–2308C at 25–458C/min was used. To determine the depletion of terpene in the HEM transport experiments, terpene standard solutions were prepared in MeOH. In the uptake experiments, terpene standard solutions were prepared in 100% ethanol.

where A0 extracted is the amount of extracted E2b. Wdry and Wwet are dry weight and wet weight of SC. Ci0 is the concentration of the E2b in aqueous 0 bulk phase. SX and S00 are the solubilities of E2b in the solution (terpene/PBS or PBS) and in PBS, respectively. The solubility ratio corrects for any activity coefficient differences between the activity coefficient in PBS and that in the terpene solution for the comparison of KE2b of the terpene solution with that of the PBS control. Partition experiments to determine the uptake amounts of the 1-octanol, 4-octanol, and 2-phenylethanol and E2b partitioning into n-heptane-treated human SC at EHEM ¼ 4 and EHEM ¼ 10 were also carried out with the same skin donors using the method described in the terpene partition experiments.

The HPLC system was consisted of two Shimadzu pumps (Kyoto, Japan), a variable wavelength UV absorbance detector, and a Sil-10A Shimadzu autoinjector with a 25 cm BDS Hypersil C18 column (Hypersil, Thermo Electron Corporation, Runcorn, UK). The detection wavelengths, mobile phases, flow rates, and retention times were: 248 nm, MeOH/water ratio of 65:35, 1.0 mL/min and 6.5 min for CS; and 280 nm, acetonitrile/water ratio of 50:50, 1.0 mL/min and 6.2 min for E2b, respectively. The standard solutions were prepared in mobile phases and were used to construct calibration curves on the basis of peak area measurement.

Partition Experiments with Delipidized Human SC

RESULTS AND DISCUSSION

The delipidized human SC was prepared according to the method described previously.27 Briefly, dried n-heptane-treated SC samples (about 3–4 mg) were weighed and transferred into 10 mL CHCl3/MeOH (2:1) mixture and equilibrated for 48 h at room

HPLC Analysis

Permeability Coefficients, Solubility Data, and Enhancement Effect Table 1 shows the permeability coefficients of CS across HEM (column 3), the CS solubility ratio (CS

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

3623

Table 1. Permeability Coefficients of Corticosterone for HEM and Corticosterone Solubility Ratio in PBS and Terpene/PBS Solutions

Terpene Menthol

Menthone

Cineole

Thymol

Carvacrol

Terpene Concentration (mM)a

Permeability Coefficient of CS for HEMb (107 cm/s)

CS Solubility Ratioc

HEM Enhancement Factor (EHEM)d

0 1.0 1.5 2.0 0 2.0 2.6 3.0 0 2.0 3.0 4.0 0 1.0 1.8 3.0 4.0 0 1.2 1.8 3.0 3.5

3.4  0.9 8.9  2.4 13.0  4.1 17.2  6.6 3.4  0.9 12.2  1.0 17.4  8.3 20.5  4.1 3.3  0.8 6.0  0.46 11.4  2.7 11.9  1.9 3.5  0.8 10.8  4.7 19.0  4.2 34.6  11.0 58  12 3.4  0.9 14.4  2.3 25.8  6.7 34.6  7.9 54  12

— 1.00  0.10 1.02  0.10 1.02  0.04 — 1.04  0.03 1.01  0.05 1.05  0.05 — 1.09  0.03 1.10  0.09 1.12  0.03 — 1.00  0.04 1.00  0.03 1.02  0.02 1.03  0.03 — 1.01  0.05 1.04  0.04 1.05  0.02 1.05  0.04

— 2.8  1.1 3.8  1.5 4.9  1.9 — 3.8  1.2 4.6  0.9 5.8  0.5 — 1.9  0.4 3.2  0.9 3.6  1.1 — 3.1  0.7 5.5  1.2 10.9  1.6 17.2  3.1 — 3.9  0.2 6.6  2.1 9.5  1.7 14.7  3.7

a Concentration of terpene in PBS (terpene/PBS) for the terpene solution. PBS alone with no terpene (0 mM terpene) was the PBS control. b Mean  SD (n  4). c Solubility ratio ¼ (CS solubility in terpene solution)/(CS solubility in PBS). d EHEM was calculated according to Eq. (1).

solubility in the terpene solution divided by CS solubility in PBS) (column 4), and the EHEM values (column 5). The enhancement factor for transport across the lipoidal pathways of HEM (EHEM) was calculated from the ratio of CS permeability coefficients in terpene/PBS to those in PBS for HEM from each human skin donor according to Eqs. (1) and (2). Thus, the HEM sample from the same human donor acted as the control to determine the enhancement factor in this experimental design. The terpene concentrations needed to induce EHEM ¼ 4 (a fourfold flux enhancement) and EHEM ¼ 10 (a tenfold flux enhancement) were then determined by interpolation in the EHEM versus terpene concentration plots (Fig. 2). The EHEM ¼ 4 and EHEM ¼ 10 concentrations are summarized in Table 2 (columns 2 and 3, respectively). For menthol, menthone, and cineole, it was not feasible to determine the EHEM ¼ 10 concentrations due to their relatively low aqueous solubilities. It was DOI 10.1002/jps

found that, except for cineole which has a higher EHEM ¼ 4 concentration than the other terpenes ( p < 0.001, ANOVA), the concentrations of EHEM ¼ 4 for thymol, carvacrol, menthol,

Figure 2. EHEM versus the concentration of terpene in terpene solution (mean  SD, n  4). Enhancement factors (EHEM) were calculated using Eq. (1). Terpene concentration is expressed in mM terpene in PBS.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3624

CHANTASART ET AL.

Table 2. Isoenhancement Concentrations (mM) of EHEM ¼ 4 and EHEM ¼ 10 Obtained with HEM for the Terpenes in PBS

Terpene

EHEM ¼ 4 Concentration (mM)a,b

EHEM ¼ 10 Concentration (mM)a

1.5  0.5 2.0  0.4 4.0  0.4 1.4  0.3 1.3  0.2

— — — 3.0  0.4 3.0  0.3

Menthol Menthone Cineole Thymol Carvacrol

*Isoenhancement concentrations are defined as the aqueous concentrations for which different enhancers induce the same extent of permeant transport enhancement, EHEM, across the lipoidal pathway of SC. a Interpolated values and interpolated uncertainties from EHEM versus enhancer concentration plots. b Using a one-way ANOVA, the EHEM ¼ 4 concentration of terpenes were significantly different. Tukey–Kramer multiple comparison tests were also used, the concentration of cineole was significantly different from other terpenes ( p < 0.001).

and menthone were not statistically different ( p > 0.05, ANOVA). Using the isoenhancement (aqueous) concentration as a measure of the enhancer potency, the aqueous EHEM ¼ 4 isoenhancement concentrations suggest that thymol, carvacrol, menthol, and menthone have similar enhancer potency. Cineole has lower enhancer potency than the other terpenes. Consistent with the EHEM ¼ 4 results, the EHEM ¼ 10 concentrations for thymol and carvacrol were found to be the same, indicating that the enhancer potencies of thymol and carvacrol were the same. HEM Recovery The CS permeability coefficients in PBS after PBS rinsing at the end of each transport experiment at

EHEM ¼ 4 and 10 concentrations, are shown in Table 3. These data show that the CS permeability coefficients in PBS after the transport experiments with the terpenes are generally greater than the permeability coefficients of the PBS control. The HEM recovery values (R) vary from 1.1 to 1.5. The relatively small increases in the permeability coefficients indicate that the exposure to the terpene solutions has caused relatively little irreversible change in the HEM barrier. The present recovery data are in agreement with the reversibility data in a previous study12 in which the authors found no significant increase in the permeability coefficient of HEM for 5-fluorouracil following terpene treatment and extensive washing.

Electrical Resistance and Permeability Coefficient of Polar Compounds Table 4 shows the electrical resistance of HEM (column 2) measured immediately after the transport experiments at the terpene concentrations of EHEM ¼ 4 and EHEM ¼ 10 for CS in the present study. The permeability coefficients of HEM for urea, Purea (in cm/s) were estimated according to the correlation between HEM permeability for urea and HEM electrical resistance:32,36,37 Log Purea ¼ Log B  Log Re

(6)

where Log B is 5.8, determined from Peck et al.,32 and Re is the HEM electrical resistance (in kV cm2). The estimated Purea are presented in the last column in Table 4. The relative low Purea under the EHEM ¼ 4 and EHEM ¼ 10 conditions of the studied terpenes is in agreement with a

Table 3. Recovery of the Terpene on Corticosterone Permeability of HEM after Transport Experiments in EHEM ¼ 4 and EHEM ¼ 10 Terpene Solutions

Terpene PBS Menthol Menthone Cineole Thymol Carvacrol

Terpenes Concentration (mM)

Permeability Coefficient of Corticosterone after Rinsing Protocola (107 cm/s)

Rb

1.5 2.0 4.0 1.4 3.0 1.3 3.0

3.5  0.8 4.2  1.5 4.8  1.5 5.1  1.6 4.3  0.8 5.2  2.5 3.7  1.7 4.7  0.9

— 1.2  0.4 1.4  0.4 1.3  0.3 1.2  0.2 1.5  0.6 1.1  0.3 1.4  0.2

a

Mean  SD (n  4). R is ratio of ( p-value of CS in PBS after rinsing protocol)/( p-value of CS in normal PBS).

b

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

3625

Table 4. Electrical Resistance and Urea Permeability Coefficient of HEM under EHEM ¼ 4 and EHEM ¼ 10 Conditions System PBS 1.5 mM 2.0 mM 4.0 mM 1.4 mM 3.0 mM 1.3 mM 3.0 mM

menthold menthoned cineoled thymold thymole carvacrold carvacrole

Log (Electrical Resistance)a,b

Permeability Coefficient of Ureaa,c (108 cm/s)

1.68  0.02 1.70  0.01 1.72  0.02 1.74  0.02 1.76  0.04 1.61  0.06 1.78  0.03 1.55  0.18

3.3  0.2 2.8  0.7 2.7  0.1 2.6  0.1 2.5  0.2 3.5  0.5 2.3  0.2 4.2  0.2

a

Mean  SD (n  4). Units of electrical resistance are kV cm2. c Permeability coefficients were estimated using Eq. (6). d EHEM ¼ 4 concentration. e EHEM ¼ 10 concentration. b

previous study27 that the contribution of the pore pathway to CS transport is negligible based on the parallel pore and lipoidal transport model and hindered transport theory. Furthermore, these results are in agreement with the previous study27 that CS is a good model steroidal permeant in studying flux enhancement across the HEM SC lipoidal pathway. Similar to the previous study,27 the electrical resistance of HEM under the enhancer conditions examined in the present study was found to be generally greater than 20 kV cm2. Therefore, the HEM barrier for polar compounds was not significantly affected by the terpenes, and the intercellular lipid remained the transport rate-determining pathway for lipophilic permeants such as CS.

Terpene Uptake into Human SC To determine terpene and permeant uptake into human SC, n-heptane-treated SC and delipidized SC were prepared. Following the procedures described in the ‘‘Partitioning Study with Human SC’’ Section, the weight percent of surface lipid of SC determined using the change in weight after nheptane treatment was found to be 13.8  7.1% (mean  SD, n ¼ 6). The weight percentage of the delipidized component of human SC determined from the weight change of the n-heptane-treated SC after CHCl3/MeOH extraction was 84.3  1.8%. This corresponds to intercellular lipid component percentage of 15.7 for the n-heptanetreated SC, and is consistent with the results obtained previously (16.4% based on n-heptanetreated human SC).27 DOI 10.1002/jps

The uptake amounts of terpenes into n-heptanetreated SC in partitioning studies under the isoenhancement conditions of EHEM ¼ 4 and EHEM ¼ 10 are presented in Table 5, column 2. The amounts of terpene in the n-heptane-treated SC were calculated by Eq. (4) using the wet and dry weights of n-heptane-treated SC as described previously.27 The amounts of terpene uptake into delipidized human SC under isoenhancement conditions of EHEM ¼ 4 and EHEM ¼ 10 (corrected for uptake into the aqueous compartment) are also presented in Table 5 (column 3). The fourth column of Table 5 shows the amounts of terpene uptake into delipidized SC normalized to the weight of n-heptane-treated human SC. The result shows that the uptake amounts of the terpene into the delipidized component of human SC are around 30–40% of those in the n-heptane-treated SC. As hypothesized in previous studies,19–21 the SC intercellular lipid enhancer concentration determined under an isoenhancement condition (e.g., EHEM ¼ 10) is related to the ‘‘intrinsic’’ enhancer potency: the lower this concentration, the more effective is the enhancer. Figure 3 presents the amount of terpene uptake into the intercellular lipids obtained by subtracting the amount of terpene uptake into the delipidized human SC (column 4 of Tab. 5) from the total amount of terpene uptake into the n-heptane-treated SC (column 2 of Tab. 5) at EHEM ¼ 4 and EHEM ¼ 10 concentrations. The data of the equilibrium uptake of the terpenes (menthol, menthone, cineole, thymol, and carvacrol) into the HEM intercellular lipids were compared with an nalkanol (i.e., 1-octanol), a branched-chain alkanol

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3626

CHANTASART ET AL.

Table 5. Terpene Uptake into n-Heptane-Treated and Delipidized Human SC under Isoenhancement Concentrations of EHEM ¼ 4 and EHEM ¼ 10 Amount of Terpene Uptake into Delipidized Human SCa

Terpene 1.5 2.0 4.0 1.4 3.0 1.3 3.0

mM mM mM mM mM mM mM

mentholc menthonec cineolec thymolc thymold carvacrolc carvacrold

Amount of Terpene Uptake into n-Heptane-Treated Human SCa Acorrected,i (mmol/mg Dry n-Heptane-Treated Human SC)

(mmol/mg Dry Delipidized Human SC)

(mmol/mg Dry n-Heptane-Treated Human SC)b

0.066  0.021 0.073  0.018 0.056  0.011 0.057  0.019 0.180  0.053 0.070  0.008 0.191  0.052

0.021  0.002 0.020  0.009 0.017  0.003 0.028  0.003 0.101  0.035 0.033  0.003 0.099  0.019

0.018  0.002 0.017  0.008 0.015  0.002 0.023  0.003 0.086  0.030 0.028  0.003 0.084  0.016

a

Mean  SD (n  4). Normalized by the weight of n-heptane-treated SC, that is, the uptake data of column 3 were multiplied by the weight percent of the delipidized component of human SC (84.3%). c EHEM ¼ 4 concentration. d EHEM ¼ 10 concentration. b

Figure 3. Amounts of the enhancer uptake from aqueous solution into the intercellular lipids and into the delipidized HEM SC. Mean  SD (n  4). A one-way ANOVA model with the studied terpenes (menthol, menthone, cineole, thymol, and carvacrol), 1-octanol, 4-octanol, and 2-phenylethanol as predictors was used to test the enhancer uptake data at EHEM ¼ 4. The Tukey–Karmer multiple comparison tests showed that no significant difference was found among the studied terpenes and alcohols ( p > 0.05) except for menthone. Only the amount of menthone uptake was higher than that of 1-octanol ( p < 0.05). A one-way ANOVA model with the thymol, carvacrol, 1-octanol, 4-octanol, and 2-phenylethanol was used to test the enhancer uptake data at EHEM ¼ 10. The Tukey–Karmer multiple comparison tests showed no significant difference of the amounts of thymol, carvacrol, 1-octanol, and 2-phenylethanol uptake into the intercellular lipids ( p > 0.05) but they were statistically lower than the 4-octanol uptake data ( p < 0.05). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

(i.e., 4-octanol) and an alkylphenyl alcohol (i.e., 2-phenylethanol). The results show that the uptake amounts (micromole/mg SC) of the terpenes and alcohols under the isoenhancement conditions of EHEM ¼ 4 into the intercellular lipids were not significantly different from each other ( p > 0.05, ANOVA) except for menthone. The uptake amounts of menthol, cineole, thymol, and carvacrol into the intercellular lipids were not significantly different from each other and from 2-phenylethanol, 4-octanol, and 1-octanol ( p > 0.05). The uptake amount of menthone was not significantly different from the other terpenes, 2-phenylethanol, and 4-octanol ( p > 0.05) but was statistically different from 1-octanol ( p < 0.05). Under the isoenhancement conditions of EHEM ¼ 10, the uptake amounts of thymol and carvacrol into the intercellular lipids were essentially the same and were not statistically different from the amounts of 1-octanol and 2-phenylethanol ( p > 0.05, ANOVA). However, the uptake amount of 4-octanol was observed to be larger than those of thymol, carvacrol, 1-octanol, and 2-phenylethanol at EHEM ¼ 10 ( p < 0.01). To conclude, these data suggest that the intrinsic potencies of the studied terpenes are not significantly different from those of n-alkanol, n-alkylphenyl alcohol, and branched-chain alkanol at EHEM ¼ 4 although there is a trend that these terpenes are less potent than the n-alkanol and n-alkylphenyl alcohol. It was also found that at EHEM ¼ 10 the intrinsic enhancer potencies of thymol and carvacrol are not significantly different from those of n-alkanol and n-alkylphenyl alcohol (although again there is

3627

a trend that the terpenes are less potent) but the potencies of these two terpenes are higher than that of branched-chain alkanol. Partitioning of E2b into Human SC The results of E2b partitioning into n-heptanetreated and delipidized human SC under the isoenhancement conditions of EHEM ¼ 4 and EHEM ¼ 10 are presented in Table 6. These partitioning coefficient data were obtained directly from the equilibrium uptake experiments and Eq. (5) as described in the Experimental Methods section. The results in the table show that around 75% and 60% of E2b was in the nonlipid domain of human SC under the EHEM ¼ 4 and EHEM ¼ 10 conditions, respectively (the delipidized SC data in column 4, Tab. 6). For PBS, approximately 85% of E2b was taken up into the SC nonlipid domain, consistent with the result in a previous study.27 As was done in the previous section for terpene uptake, the KE2b values for the intercellular lipids was obtained by subtracting the normalized partition coefficient component of KE2b determined in the experiments with delipidized SC (column 4, Tab. 6) from the data of n-heptane-treated SC (column 2, Tab. 6). The KE2b results of human SC intercellular lipids are summarized in Figure 4. The KE2b results obtained with 1-octanol, 2-phenylethanol, and 4-octanol under the same conditions (EHEM ¼ 4 and EHEM ¼ 10 conditions) are also shown in Figure 4. It can be seen that within the data scatter, the enhancement of the E2b partitioning

Table 6. Partition Coefficient of b-Estradiol (KE2b) for n-Heptane-Treated and Delipidized Human SC under Isoenhancement Concentrations of EHEM ¼ 4 and EHEM ¼ 10 Partition Coefficient of E2ba Terpene PBS 1.5 mM 2.0 mM 4.0 mM 1.4 mM 3.0 mM 1.3 mM 3.0 mM

menthold menthoned cineoled thymold thymole carvacrold carvacrole

n-Heptane-Treated Human SCb

Delipidized Human SCb

Delipidized Human SCc

134  23 153  15 149  9 147  19 154  14 229  25 151  23 228  25

134  22 135  20 131  29 138  10 134  4 162  13 134  22 163  27

113  19 114  17 110  25 116  9 114  3 136  11 113  19 137  22

a

Mean  SD (n  4). Corrected for the partition coefficient into the aqueous compartment using Eq. (5). c Further corrected to the n-heptane-treated Human SC; the weight of dry n-heptane-treated human SC decreased by 15.7% after delipidization treatment. d EHEM ¼ 4 concentration. e EHEM ¼ 10 concentration. b

DOI 10.1002/jps

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3628

CHANTASART ET AL.

Figure 4. Partition coefficients of estradiol (KE2b) for partitioning from aqueous solution into the intercellular lipids and into the delipidized HEM SC. Mean  SD (n  4). A one-way ANOVA model with the studied terpenes (menthol, menthone, cineole, thymol, and carvacrol), 1-octanol, 4-octanol, and 2-phenylethanol as predictors was used to test the KE2b data at EHEM ¼ 4. No significant difference was found among the studied terpenes and alcohols ( p > 0.05). A one-way ANOVA model with thymol, carvacrol, 1-octanol, 4-octanol, and 2-phenylethanol as predictors was used to test the KE2b data at EHEM ¼ 10. The Tukey–Karmer multiple comparison tests showed that the amount of 4-octanol uptake was significantly different from thymol, carvacrol, 1-octanol, and 2-phenylethanol ( p < 0.05).

into the intercellular lipids induced by the studied terpenes, 1-octanol, 4-octanol, and 2-phenylethanol all fall in the same range when compared under the same EHEM conditions. The E2b partitioning enhancement was around twofold and five- to sevenfold at EHEM ¼ 4 and EHEM ¼ 10 conditions, respectively. The E2b partitioning enhancement results at EHEM ¼ 10 are consistent with those observed previously in HEM27 and HMS19,21. It was also noticed from the same terpene enhancers at EHEM ¼ 4 and EHEM ¼ 10 a correlation between terpene uptake into the intercellular lipid SC and E2b partitioning enhancement: the more terpene uptake, the higher E2b partitioning enhancement. This indicates that terpene enhancer concentration in the intercellular lipid can promote drug partitioning. Terpene Partitioning into SC Intercellular Lipids and Terpene Octanol-PBS Partition Coefficient In a previous study,27 the plot of the logarithm of the aqueous-to-SC partition coefficients

(Kintercellular lipid/PBS) and the logarithm of octanol-PBS partition coefficient (Koctanol/PBS) of 1-hexanol, 1-heptanol, 1-octanol, 2-phenylethanol and 1-octyl-2-pyrrolidone was examined. The linear relationship between Kintercellular lipid/PBS and Koctanol/PBS with a slope close to unity suggested that the microenvironment of the enhancers was well-mimicked by liquid octanol. In the present study, Kintercellular lipid/PBS were calculated from the terpene uptake data and isoenhancement aqueous concentration at EHEM ¼ 4. The log Kintercellular lipid/PBS are 1.39  0.22, 1.51  0.08, 1.51  0.12, 1.45  0.12, and 1.01  0.09, and the logarithm of octanol– water partition coefficient (Log Koctanol/water)38 are 3.52, 3.52, 3.38, 2.87, and 3.13 for thymol, carvacrol, menthol, menthone, and cineole, respectively. Within the data scatter, the values of Kintercellular lipid/PBS for the terpenes fall in the same range as those of 1-octanol and 4-octanol in the same Koctanol/water range. The results suggest that the microenvironment of the studied terpenes is also well-mimicked by liquid octanol.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

Insights into Terpenes as Skin Permeation Enhancers Terpenes, neat or combination with cosolvents (e.g., ethanol or propylene glycol) have been investigated as skin permeation enhancers for both lipophilic and hydrophilic drugs.15,39,40 Due to the different experimental designs employed in the permeation studies by various research groups, the skin permeation enhancing mechanisms of terpenes concluded in these studies show discrepancies. For example, Williams and Barry12,15 investigated the effectiveness of alcohols, ketones, and oxides terpenes as skin permeation enhancers and found that the oxygencontaining terpenes (carveol and menthone) were not effective in enhancing the permeation of a lipophilic drug (estradiol) but effective in enhancing that of a hydrophilic drug (5-fluorouracil). Only the cyclic oxide terpenes (1,8-cineole) were found to be effective in enhancing lipophilic drug penetration in their studies. In addition, the fluxenhancing mechanism of the terpenes was shown to occur mainly by increasing the diffusivity of the drug through the SC. El-Kattan et al.41 also found that thymol was more effective at enhancing the penetration of hydrophilic drugs than lipophilic drugs in their examination of terpenes on model drugs with different lipophilicities. In a study conducted by Jain et al.42 on the effects of menthol, cineole, and menthone upon the hydrophilic permeant imipramine hydrochloride (IMH), it was proposed that the contribution of enhanced diffusivity in the permeation of IMH was higher in comparison to that of partitioning with the terpene treatment. Fourier transform infrared spectra of epidermal membrane treated with menthol, cineole and menthone in ethanol/water (2:1) further supported this mechanism of permeation enhancement. On the other hand, for the permeation of tamoxifen, Zhao and Singh43 suggested that the flux enhancement induced by menthone is due to lipid extraction and not by improving the partitioning of the lipophilic drug into the SC. Narishetty and Panchagnula14 also investigated the effects of cineole, menthol, and menthone on the permeation of hydrophilic zidovudine across rat skin and came to a similar conclusion. It was suggested that the terpenes interacted with the SC lipid components. In addition, Stott et al.44 investigated the transdermal delivery of ibuprofen-terpene (thymol, menthol, and cineole) eutectic systems and suggested the involvement of hydrogen bond interactions between the drug and terpenes in DOI 10.1002/jps

3629

drug permeation. Yamane et al.45 concluded that the mechanism of action of cineole and menthone in PG/water involved SC lipid disruption in their differential scanning calorimetry study. In the present study, the effects of various oxygen-containing terpenes (menthol, thymol, carvacrol, menthone, and cineole) on the permeation of a lipophilic drug (CS, that predominantly utilizes the SC lipoidal pathway) were investigated in an aqueous system without any cosolvents under one single experimental condition. Together with the results of the relative high electrical resistance of HEM observed in the present study, the present HEM transport data suggest that the oxygen-containing terpenes are mainly effective in enhancing the permeation of lipophilic compounds. In addition, the human SC partitioning results of the terpenes, compared with an n-alkanol (i.e., 1-octanol), a branchedchain alkanol (i.e., 4-octanol) and an n-alkylphenyl alcohol (i.e., 2-phenylethanol) and the conclusions in the previous HMS and HEM studies,19,23,27 suggest that the intrinsic enhancer potencies of the studied terpenes and those of nalkanols and an alkylphenyl alcohol are essentially the same and better than that of the branched-chain alkanols. The oxygen-containing terpenes as a chemical enhancer class do not show a large deviation from the hypotheses made in these previous studies. This also suggests that the oxygen-containing terpenes enhance the permeability of lipophilic drugs mainly by SC intercellular lipid fluidization. The conclusions in our present and previous studies differing from those in other research groups studying the terpenes (and other chemical permeation enhancers) may be related to the different experimental designs employed in the studies. Particularly, a major difference was that the present study employed a symmetric and equilibrium experimental condition in regard to the aqueous enhancer solution in both the donor and receiver chambers—the enhancer was present in both the donor and receiver chambers and was in equilibrium with the SC.46,47 Second, there was no cosolvent involved in the present study and this negated any potential synergistic effects from the cosolvent on the terpenes. The absence of cosolvent allowed the mechanistic evaluation of the direct effects and the quantitation of the enhancer potencies of the terpenes as a cosolvent would make data interpretations in the enhancer studies difficult. For example, it has been suggested that the application of terpenes (cineole and menthone)

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3630

CHANTASART ET AL.

in propylene glycol (PG)/water cosolvent systems significantly enhanced the permeation of 5-fluorouracil (a hydrophilic compound) and the permeation enhancement activities of the terpenes were related to the PG content in the vehicles.45 In other studies, the presence of high amounts of ethanol (50%), either as a cosolvent or a composition in drug formulations, was also found to play an important role in promoting drug diffusivity across the skin.41,42 Enhancer uptake into SC under different enhancement conditions has been used to evaluate the mechanisms of terpenes as permeation enhancers by various research groups. Similar to the problem encountered in the permeation studies, the interactions between terpenes and SC in the presence of cosolvents may be different from those without, and this may affect enhancer uptake into SC. For example, Kobayashi et al.48 reported that adding ethanol in the donor solution increased the migration of menthol into SC. Mackay et al.49 also found that increasing the PG content of cosolvent systems increased the amount of menthol taken up by SC. Moghimi et al.50 proposed that cineole increased the partitioning of 5-fluorouracil into the intercellular lipids via complexation and thereby increased the permeation of drugs by a form of facilitated transport. Such interactions between terpenes and cosolvents in SC are difficult to predict and interpret due to the differences in the physicochemical characteristics of terpenes and the amounts of cosolvents used in these studies. The present investigation has also avoided this problem.

CONCLUSION The present study was aimed at (a) assessing the relative effectiveness of various oxygen-containing terpenes (menthol, thymol, carvacrol, menthone, and cineole) as skin permeation enhancers on drug transport across the SC lipoidal pathway, (b) determining the environment of the site of enhancer action of the terpenes in HEM, (c) testing the previous hypotheses with a new class of chemical enhancers—oxygen-containing terpenes—and (d) gaining additional insights into the skin permeation enhancing mechanisms of the terpenes. The results in the transport experiments suggest that the terpenes are effective in enhancing the permeation of lipophilic compounds. The enhancer potencies of menthol,

thymol, carvacrol, and menthone are essentially the same and higher than that of cineole based on their aqueous concentration in the diffusion cell chamber. The intrinsic potencies of the terpenes are similar to those of the n-alkanol and the n-alkylphenyl alcohol and higher than those of the branched-chain alkanol based on their concentration in the SC, suggesting that the mechanisms of action of the oxygen-containing terpenes are similar to the previously studied enhancers such as the n-alkyl alcohols. The E2b data are consistent with drug partitioning enhancement being a major mechanism of terpenes to enhance skin permeation. The Kintercellular lipid/PBS versus Koctanol/water values for the terpenes fall in the same range as those of 1-octanol and 4-octanol. This suggests that the microenvironment of the studied terpenes is well-mimicked by liquid noctanol. The findings in the present study for the oxygen-containing terpenes, within the variability of the data, are generally consistent with those observed previously, which continues to support the hypotheses made in our previous studies19–21,27 on the mechanism of action of permeation enhancers.

ACKNOWLEDGMENTS The financial supports of the Thailand Research Fund (MRG 5080136) and the Commission on Higher Education, Ministry of Education, Thailand are gratefully acknowledged. The authors thank Yanhee General Hospital for kindly supplying us the human skin samples. The authors also thank Dr. Kittisak Sripha for helpful discussion.

REFERENCES

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

1. Barry BW. 1991. Lipid-protein-partition theory of skin penetration enhancement. J Control Release 15:237–248. 2. Ogiso T, Iwaki M, Paku T. 1995. Effect of various enhancers on transdermal penetration of indomethacin and urea, and relationship between penetration parameters and enhancement factors. J Pharm Sci 84:482–488. 3. Sinha VR, Kaur MP. 2000. Permeation enhancers for transdermal drug delivery. Drug Dev Ind Pharm 26:1131–1140. 4. Williams AC, Barry BW. 1992. Skin absorption enhancers. Crit Rev Ther Drug Carr Sys 9:305–353. DOI 10.1002/jps

OXYGEN-CONTAINING TERPENES AS SKIN PERMEATION ENHANCERS

5. Aqil M, Ahad A, Sultana Y, Ali A. 2007. Status of terpenes as skin penetration enhancers. Drug Discov Today 12:1061–1067. 6. Kanikkannan N, Kandimalla K, Lamba SS, Singh M. 2000. Structure-activity relationship of chemical penetration enhancers in transdermal drug delivery. Curr Med Chem 7:593–608. 7. Va´vrova´ K, Zbytovska´ J, Hraba´lek A. 2005. Amphiphilic transdermal permeation enhancers: Structureactivity relationships. Curr Med Chem 12:2273– 2291. 8. Walker R, Smith EW. 1996. The role of percutaneous penetration enhancers. Adv Drug Deliv Rev 18:295–301. 9. Thakur RA, Wang Y, Michniak B. 2006. Essential oils and terpenes. In: Smith EW, Maibach HI, editors. Percutaneous penetration enhancers, 2nd edition. Florida: CRC Press. pp 159–173. 10. Obata Y, Takayama K, Machida Y, Nagai T. 1991. Combined effect of cyclic monoterpenes and ethanol on percutaneous absorption of diclofenac sodium. Drug Des Discov 8:137–144. 11. Okabe H, Obata Y, Takayama K, Nagai T. 1990. Percutaneous absorption enhancing effect and skin irritation of monocyclic monoterpenes. Drug Des Deliv 6:229–238. 12. Williams AC, Barry BW. 1991. Terpenes and the lipid-protein-partitioning theory of skin penetration enhancement. Pharm Res 8:17–24. 13. Amnuaikit C, Ikeuchi I, Ogawara K, Higaki K, Kimura T. 2005. Skin permeation of propranolol from polymeric film containing terpene enhancers for transdermal use. Int J Pharm 289:167– 178. 14. Narishetty STK, Panchagnula R. 2004. Transdermal delivery of zidovudine: Effect of terpenes and their mechanism of action. J Control Release 95: 367–379. 15. Williams AC, Barry BW. 1991. The enhancement index concept applied to terpene penetration enhancers for human skin and model lipophilic (estradiol) and hydrophilic (5-fluorouracil) drugs. Int J Pharm 74:157–168. 16. Arellano A, Santoyo S, Martin C, Ygartua P. 1996. Enhancing effect of terpenes on the in vitro percutaneous absorption of diclofenac sodium. Int J Pharm 130:141–145. 17. Kararli TT, Kirchhoff CF, Penzotti SC. 1995. Enhancement of transdermal transport of azidothymidine (AZT) with novel terpene and terpene-like enhancers: In vivo-in vitro correlations. J Control Release 34:43–51. 18. Okabe H, Takayama K, Ogura A, Nagai T. 1989. Effect of limonenes and related compounds on the percutaneous absorption of indomethacin. Drug Des Deliv 4:313–321. 19. Chantasart D, Li SK, He N, Warner KS, Prakongpan S, Higuchi WI. 2004. Mechanistic studies of

DOI 10.1002/jps

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

3631

branched-chain alkanols as skin permeation enhancers. J Pharm Sci 93:762–779. He N, Li SK, Suhonen TM, Warner KS, Higuchi WI. 2003. Mechanistic study of alkyl azacycloheptanones as skin permeation enhancers by permeation and partition experiments with hairless mouse skin. J Pharm Sci 92:297–310. He N, Warner KS, Chantasart D, Shaker DS, Higuchi WI, Li SK. 2004. Mechanistic study of chemical skin permeation enhancers with different polar and lipophilic functional groups. J Pharm Sci 93:1415– 1430. Kim YH, Ghanem AH, Mahmoud H, Higuchi WI. 1992. Short chain alkanols as transport enhancers for lipophilic and polar/ionic permeants in hairless mouse skin mechanism(s) of action. Int J Pharm 80:17–31. Warner KS, Li SK, He N, Suhonen TM, Chantasart D, Bolikal D, Higuchi WI. 2003. Structureactivity relationship for chemical skin permeation enhancers: Probing the chemical microenvironment of the site of action. J Pharm Sci 92:1305– 1322. Yoneto K, Li SK, Higuchi WI, Shimabayashi S. 1998. Influence of the permeation enhancers 1-alkyl-2-pyrrolidones on permeant partitioning into the stratum corneum. J Pharm Sci 87:209–214. Yoneto K, Ghanem AH, Higuchi WI, Peck KD, Li SK. 1995. Mechanistic studies of the 1-alkyl-2-pyrrolidones as skin permeation enhancers. J Pharm Sci 84:312–317. He N, Warner KS, Higuchi WI, Li SK. 2005. Model analysis of flux enhancement across hairless mouse skin induced by chemical permeation enhancers. Int J Pharm 297:9–21. Chantasart D, Sa-Nguandeekul P, Prakongpan S, Li SK, Higuchi WI. 2007. Comparison of the effects of chemical permeation enhancers on the lipoidal pathways of human epidermal membrane and hairless mouse skin and the mechanism of enhancer action. J Pharm Sci 96:2310–2326. Lund W. 1994. Buffered solutions. In: Lund W, editor. The pharmaceutical codex, 12th edition. London: The Pharmaceutical Press. pp 66–68. Klingman AM, Christophers E. 1963. Preparation of isolated sheets of human stratum corneum. Arch Dermatol 88:702–705. Bronaugh RL, Collier SW. 1993. In vitro methods for measuring skin permeation. In: Zatz JL, editor. Skin permeation fundamentals and application. Wheaton, IL: Allured Publishing Corp. pp 93–111. Kasting GB, Bowman LA. 1990. DC electrical properties of frozen, excised human skin. Pharm Res 7:134–143. Peck KD, Ghanem AH, Higuchi WI. 1995. The effect of temperature upon the permeation of polar and ionic solutes through human epidermal membrane. J Pharm Sci 84:975–982.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

3632

CHANTASART ET AL.

33. Warner KS, Li SK, Higuchi WI. 2001. Influences of alkyl group chain length and polar head group on chemical skin permeation enhancement. J Pharm Sci 90:1143–1153. 34. Ghanem AH, Mahmaud H, Higuchi WI, Rohr U, Borsadia S, Lui P, Fox JL, Good WR. 1987. The effects of ethanol on the transport of b-estradiol and other permeants in hairless mouse skin. II. A new quantitative approach. J Control Release 6:75– 83. 35. Raykar PV, Fung MC, Anderson BD. 1988. The role of protein and lipid domains in the uptake of solutes by human stratum corneum. Pharm Res 5:140– 150. 36. Li SK, Ghanem AH, Peck KD, Higuchi WI. 1998. Characterization of the transport pathways induced during low to moderate voltage iontophoresis in human epidermal membrane. J Pharm Sci 87:40–48. 37. Li SK, Ghanem AH, Peck KD, Higuchi WI. 1999. Pore induction in human epidermal membrane during low to moderate voltage iontophoresis: A study using AC iontophoresis. J Pharm Sci 88:419–427. 38. Interactive LogKow (KowWin) Demo http://www. syrres.com/esc/est_kowdemo.htm (accessed March, 2008). 39. Ghafourian T, Zandasrar P, Hamishekar H, Nokhodchi A. 2004. The effect of penetration enhancers on drug delivery through skin: A QSAR study. J Control Release 99:113–125. 40. Hori M, Satoh S, Maibach HI, Guy RH. 1991. Enhancement of propranolol hydrochloride and diazepam skin absorption in vitro: Effect of enhancer lipophilicity. J Pharm Sci 80:32–35. 41. EL-Kattan A, Asbill C, Michniak B. 2000. The effect of terpene enhancer lipophilicity on the percutaneous permeation of hydrocortisone formulated in HPMC gel systems. Int J Pharm 198:179–189.

42. Jain AK, Thomas NS, Panchagnula R. 2002. Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes. J Control Release 79:93–101. 43. Zhao K, Singh J. 1998. Mechanisms of percutaneous absorption of tamoxifen by terpenes: Eugenol, D-limonene and menthone. J Control Release 55:253–260. 44. Stott PW, Williams AC, Barry BW. 1998. Transdermal delivery from eutectic systems: Enhanced permeation of a model drug, ibuprofen. J Control Release 50:297–308. 45. Yamane MA, Williams AC, Barry BW. 1995. Terpene penetration enhancers in propylene glycol/ water co-solvent systems: Effectiveness and mechanism of action. J Pharm Pharmacol 47: 978–989. 46. Li SK, Higuchi WI. 2006. Quantitative structureenhancement relationship and the microenvironment of the enhancer site of action. In Smith EW, Maibach HI, editors. Percutaneous penetration enhancers, 2nd edition. Boca Raton, FL: CRC Press. pp 35–49. 47. Li SK, Higuchi WI. 2006. Mechanistic studies of permeation enhancers. In Smith EW, Maibach HI, editors. Percutaneous penetration enhancers. 2nd edition. Boca Raton, FL: CRC Press. pp 271–292. 48. Kobayashi D, Matsuzawa T, Sugibayashi K, Morimoto Y, Kimura M. 1994. Analysis of the combined effect of 1-menthol and ethanol as skin permeation enhancers based on a two-layer skin model. Pharm Res 11:96–103. 49. Mackay KM, Williams AC, Barry BW. 2001. Effect of melting point of chiral terpenes on human stratum corneum uptake. Int J Pharm 228:89–97. 50. Moghimi HR, Williams AC, Barry BW. 1998. Enhancement by terpenes of 5-fluorouracil permeation through the stratum corneum: Model solvent approach. J Pharm Pharmacol 50:955–964.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 10, OCTOBER 2009

DOI 10.1002/jps