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Effect of unsaturated menthol analogues on the in vitro penetration of 5-fluorouracil through rat skin
International Journal of Pharmaceutics 443 (2013) 120–127
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Effect of unsaturated menthol analogues on the in vitro penetration of 5-fluorouracil through rat skin Yang Chen a , Jian Wang a , Dongmei Cun a , Manli Wang a,b , Juan Jiang a , Honglei Xi a , Hongxia Cui a , Yongnan Xu a , Maosheng Cheng a , Liang Fang a,∗ a b
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, People’s Republic of China School of Pharmacy, Beihua University, 3999 Huashan Road, Jilin, Jilin 132013, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 16 October 2012 Accepted 7 January 2013 Available online 16 January 2013 Keywords: Transdermal Absorption enhancer Unsaturated Menthol analogue Mechanism Structure–activity relationship
a b s t r a c t To explore the structure–activity relationship for terpenes as transdermal penetration enhancers, unsaturated menthol analogues were synthesized in our study, including p-menth-1-en-3-ol (Compd 1), p-menth-4-en-3-ol (Compd 2), p-menth-4(8)-en-3-ol (Compd 3) and p-menth-8-en-3-ol (Compd 4). Their enhancing activity on the penetration of 5-fluorouracil through rat skin was evaluated by in vitro experiments. Attenuated total reflection-Fourier transform infrared spectroscopy, molecular modeling and transepidermal water loss (TEWL) were introduced to investigate the enhancer induced alteration in different skin lipid domains. The results indicated that Compd 3 achieved the highest enhancement ability with an enhancement ratio of 3.08. Other analogues were less effective than Compd 3, and no significant difference was found between them and menthol. Treatment of rat skin with these enhancers did not produce any shift in the stretching vibration of the methylene in hydrophobic lipid chains, but significantly improved the polar pathway across the rat skin as suggested by the increased TEWL. Molecular modeling results suggested that polar head groups of the skin lipids provided the main binding site for enhancer action. These findings indicated that the studied compounds enhanced drug transport by interacting with the polar domain of the skin lipid, instead of by affecting the arrangement of the hydrophobic chains. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The outermost layer of the skin, stratum corneum (SC), provides the principal rate limitation to the diffusion of drugs and water across the skin (Hadgraft, 2004). It is generally accepted that this barrier function is strongly associated with the complex and orderly organized structure of the intercellular lipids in SC (Wertz and van den Bergh, 1998; Menon, 2002). To enhance the skin permeability, many approaches have been used, including the chemical and physical techniques (Barry, 2001). The chemical approach has led to the introduction of transdermal penetration enhancers, which can be used to facilitate the percutaneous absorption by temporarily diminishing the impermeability of the skin (Finnin and Morgan, 1999; Jain et al., 2006). Terpenes represent one of the promising candidates for clinically acceptable enhancers (Aqil et al., 2007). Depending on the presence of carbon-carbon double bond, they can be classified as two types, i.e., saturated and unsaturated terpenes. Quantitative
∗ Corresponding author. Tel.: +86 24 23986330; fax: +86 24 23986330. E-mail address: [email protected] (L. Fang). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.015
structure–activity relationship (QSAR) study has revealed that the activity of terpenes is primarily related to their chemical structures (Ghafourian et al., 2004; Iyer et al., 2007; Kang et al., 2007), and the unsaturated terpenes seem to be better candidates than the saturated ones in promoting the transdermal absorption of 5fluorouracil (5-FU) (Kang et al., 2007). However, the inter laboratory difference and the large structural diversity of terpenes in the QSAR models might lead to a misjudgment of the result. Therefore, a detailed structural analysis about the terpenes is still needed to describe a precise structure–activity relationship. The present study was carried out to investigate the exact effect of double bond on the enhancement ability of the unsaturated terpenes. Menthol (MT), a saturated terpene that has widely been used as a penetration enhancer, was selected as the lead compound. A series of unsaturated analogues were obtained by introduction of a double bond onto its four different molecular positions, which gave the product of p-menth-1-en-3-ol (Compd 1), p-menth-4-en-3-ol (Compd 2), p-menth-4(8)-en-3-ol (Compd 3) and p-menth-8-en3-ol (Compd 4). Their enhancement effect on the transdermal permeability of a hydrophilic drug, 5-FU, was studied by comparison with MT. The structures of the drug, MT and its analogues were illustrated in Fig. 1. The strong structural similarity between MT
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Fig. 1. Chemical structures and molecular models of 5-FU, MT and its analogues. Carbon atoms were colored gray, oxygen atoms red, nitro blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and its unsaturated analogues made it possible to explore whether the double bond in unsaturated compounds could influence their transdermal promoting effect. One the other hand, although many studies have investigated the transdermal enhancement effects of terpenes, their exact mechanisms for action are not clearly defined (Chantasart et al., 2009). Data about the molecular interaction between SC lipids and enhancers, which plays an important role in understanding the mechanisms of enhancement, is still lacking so far. Previous studies have proposed that the effect of a penetration enhancer on the lipid bilayer may involve interactions at two sites, namely the polar head groups and the hydrophobic tails of the intercellular lipid (Barry, 1991). Therefore, in the present study, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was introduced to study the local effect of enhancers on the hydrophobic tails of the intercellular lipids (Guillard et al., 2009). Molecular modeling was used to observe the molecular interaction between enhancers and the polar head groups in SC lipids (Narishetty and Panchagnula, 2004). Finally, transepidermal water loss (TEWL) was selected as a noninvasive bioengineering method to investigate the possible alteration in polar pathway after skin treatment with different enhancers (Levin and Marbach, 2005). The objectives of the present study were to (a) determine the structure–enhancement relationship for MT and its unsaturated analogues, (b) respectively investigate the alterations in the
(1) Compd 1
hydrophobic moieties and polar domain of SC lipid after skin treatment with different enhancers, and (c) correlate these structural or functional changes with the enhancement activity towards 5-FU. 2. Materials and methods 2.1. Materials Compd 1, Compd 2 and Compd 3 were synthesized in our laboratory. Compd 4, piperitone and menthone were bought from TCI (Tokyo, Japan). Pulegone was obtained from Alfa Aesar (Ward Hill, MA, USA). 2-Isopropyl-5-methylcyclohexyl heptanoate (M-HEP) was prepared as described previously (Zhao et al., 2008). Isopropyl myristate (IPM) and MT were both purchased from China National Medicines Co., Ltd. (Shanghai, China). 5-FU was provided by Fangge Pharmaceutical Co., Ltd. (Zhejiang, China). Methanol of HPLC grade was supplied by the Yuwang Pharmaceutical Co., Ltd. (Shandong, China). All of other chemical reagents were of at least reagent grade available. 2.2. Synthesis of unsaturated MT analogues As shown in Fig. 2a and b, Compd 1 and Compd 3 were respectively prepared from piperitone and pulegone by LiAlH4 selective reduction (Serra et al., 2003). Briefly, a solution of corresponding substrate (5 g, 32.85 mmol) in absolute diethyl ether (15 ml) was
(2) Compd 2 a
a O
OH
O
OH
(3) Compd 3 b O
c Br
O
a O
OH
Fig. 2. Synthetic methods of Compd 1 (1), Compd 3 (2), and Compd 2 (3). a: LiAlH4 /Et2 O; b: N-bromosuccinide/benzoyl peroxide/CCl4 ; c: quinoline.
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added dropwise under nitrogen into a stirred equimolar suspension of LiAlH4 in absolute diethyl ester (50 ml) at 0 ◦ C. The temperature of the reaction was also kept at 0 ◦ C. After agitation for 2.5 h, the reaction was terminated by dropwise addition of water and 10% aqueous solution of NaOH. Then, the ether layer was separated, washed with brine, dried with anhydrous Na2 SO4 , and freed of volatile solvent under vacuum. The residue was purified by silica gel column chromatography with gradient elution consisting of ethyl acetate and petroleum ether (1:100→20:80) to give pure Compd 1 (yield, 82%), 1 H NMR (300 MHz, DMSO, ı): 0.752 and 0.894 (dd, J = 6.9 Hz, 6H, –CH(CH3 )2 ), 1.120–1.149 (m, 2H, –CH2 ), 1.364–1,384 (m, 1H, –CH), 1.602 (s, 3H, –CH3 ), 1.851–1.964 (m, 2H, –CH2 ), 1.964–2.057 (m, 1H, –CH), 3.774 (br, s, 1H, OH), 4.414 (d, J = 6.3 Hz, 1H, –O CH), 5.289 (s, 1H, CH); ESI-MS m/z: 154 (M+ ) and Compd 3 (yield, 78%), 1 H NMR (300 MHz, DMSO, ı): 0.912 (d, J = 6.3 Hz, 3H, –CH3 ), 1.155 (s, 6H, C(CH3 )2 ), 1.514–1.719 (m, 4H, –CH2 , –CH2 ), 1.972–2.130(m, 3H, –CH2 , –CH), 4.280 (s, 1H, –O CH), 5.582 (br, s, 1H, –OH); ESI-MS m/z: 154 (M+ ). The purity of Compd 1 and Compd 3 was over 95% as characterized by GC/MS. The synthetic sequence for Compd 2 was outlined in Fig. 2c. N-Bromosuccinide (8.4 g, 47.20 mmol) and benzoyl peroxide (1 g) were added to menthone (6 g, 32.91 mmol) which was dissolved in carbon tetrachloride (50 ml). The reaction mixture was then heated to reflux for 2 h while stirring. After being cooled and filtered, the obtained insoluble was washed with carbon tetrachloride, and the collected filtrates were washed successively with water, 10% sodium carbonate solution, water and brine. The solvent was removed under vacuum, and the residue was taken up in quinoline (11 g) and heated at 150 ◦ C for 30 min. The corresponding ketone product was afforded after a simple purification by chromatography eluting with ethyl acetate and petroleum ether (1:200→20:80) (Paquette and Doehner, 1980). The following step was performed in accordance with the synthetic scheme of Compd 1 and Compd 3, giving pure product of Compd 2 (yield, 50%), 1 H NMR (300 MHz, DMSO, ı): 0.902 (d, J = 7.5 Hz, 3H, –CHCH ), 3 0.937–0.983 (t, J = 6.9 Hz, 6H, –CH(CH3 )2 ), 1.135–1.174 (m, 1H, –CH), 1.557–1.606 (m, 2H, –CH2 ), 1.853–1.983 (m, 2H, –CH2 ), 2.502–2.623 (m, 1H, –CH), 4.519 (d, J = 6.6 Hz, 1H, –OCH), 5.290 (d, J = 4.2 Hz, 1H, CH); ESI-MS m/z: 154 (M+ ). The purity of Compd 2 was over 95% as characterized by GC/MS.
the guidelines for animal use published by the Life Science Research Center of Shenyang Pharmaceutical University. 2.3.3. In vitro permeation experiments Skin permeation experiments were performed according to the method of Fang et al. (2002). In vitro skin penetration experiments were carried out at 32 ◦ C in modified two-chamber diffusion cells. Each half-cell had a volume of 3 ml and an available diffusion area of 0.95 cm2 . The skin fragments were mounted onto the cells with the SC in contact with the donor chamber. The acceptor chamber was filled with 3 ml distilled water, and the donor chamber with drug suspension. Both compartments were continuously stirred with star-head bars at 600 rpm throughout the experiments. Samples (2 ml) from the acceptor chambers were withdrawn at predetermined intervals over 8 h, and analyzed by HPLC with the calibration of a standard curve. At each time point, the withdrawn sample was replaced with an equivalent volume of acceptor liquid. The experiments were performed in quadruplicates. 2.3.4. HPLC analysis of the drug The drug content was analyzed with an HPLC system consisting of an L-2420 variable-wavelength ultraviolet absorbance detector and an L-2130 pump (Hitachi High-Technologies Corporation, Tokyo, Japan). The reversed phase stainless-steel column (20 cm × 4.6 mm) was packed with Diamonsil C-18 (5 m particle size; Dikma Technologies, Beijing, China). The column temperature was maintained at 40 ◦ C. The eluent was detected at 265 nm. The mobile phase was composed entirely of distilled water and the flow rate was 1 ml min−1 (United States Pharmacopeia, 2009). 2.3.5. Data analysis The cumulative amount of penetrated drugs per unit area across the skin in 8 h (Q8 h , g cm−2 ) was calculated according to the measured drug concentration in receptor-phase after being corrected for sampling effects. The steady state flux (Js , g cm−2 h−1 ) and lag time (Tlag , h) were obtained from the slope and the x-intercept of the linear portion of the profile plotted between Q8 h on Y-axis and time on the X-axis. In order to normalize the promoting effect of different penetration enhancers, enhancement ratio (ERQ and ERflux ) was defined as follows: Q8 h (with enhancer) Q8 h (without enhancer)
(1)
Js (with enhancer) Js (without enhancer)
(2)
2.3. In vitro skin penetration experiments
ERQ =
2.3.1. Preparation of donor solutions Donor solutions were prepared by dissolving 5% (w/w) enhancers and excess amounts of 5-FU in IPM. Then the suspensions were stirred for 5 min, followed by sonication for 10 min and equilibration overnight. Control sample was obtained in a similar way without addition of any enhancers.
ERflux =
2.3.2. Skin preparation Full-thickness skins were gained from Male Wistar rats (180–220 g, 6–8 weeks old), which were provided by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). After the rats were anesthetized with urethane (20%, w/v, i.p.), the abdominal hair was removed by an electric clipper, and then carefully shaved with a razor. Full-thickness skins (i.e. epidermis with SC and dermis) were excised from the shaved abdomen sites of the sacrificed rats. The adhering subcutaneous fat and other extraneous tissue were surgically trimmed. After being washed and examined for integrity under the microscope, the skin was wrapped in aluminium foil and then subsequently stored at −70 ◦ C for a maximum of 2 weeks. The skins were allowed to thaw slowly prior to use in order to avoid damage. All the animal procedures were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and also in accordance with
2.4. Determination of drug solubility in donor solution An excessive amount of drug was added to the donor solution with or without enhancers. The mixture was shaken vigorously, and then allowed to equilibrate at 32 ± 1 ◦ C in a shaken water-bath for 48 h. Finally the suspension was filtered through a 0.45-m Millipore filter and analyzed by HPLC after appropriate dilution. The experiments were performed in quadruplicates. 2.5. ATR-FTIR experiment The rats were anesthetized with urethane (20%, w/v, i.p.) and the abdominal hair was shaved as mentioned above. Then the rat abdomen was divided into 4 different sections with each area of 1 cm2 . Ten microliter each enhancer solution (10 mg dissolved in 100 l ethanol) was applied onto the marked areas for an exposure time of 30 min. Skin treated with solvent alone was determined as control. After treatment, the skin was blotted with tissue paper and dried at room temperature for 15 min. Each area of diffusion was then subjected to ATR-FTIR analysis (NEXUS+70, Thermo Electron
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Corporation, MA, USA). ATR-FTIR spectra were recorded at 2 cm−1 resolution. 2.6. Molecular modeling Eight-times repeat units of ceramide NP (Cer NP) were built in three-dimensional (3D) coordinates by means of Sybyl 6.91 software package (Tripos, Inc., St. Louis, MO, USA). After addition of all the hydrogen atoms with Amber 7 FF99 force field, partial charges were assigned to Cer NP molecules according to the method of Gastei-ger-Hückel. The structures were subjected to further energy minimization using both steepest descent and conjugate gradient protocols. Docking calculations were preformed on AutoDock 3.05 (the Scripps Research Institute, La Jolla, FL, USA). The fully flexible 5FU, MT and its analogues, were built in 3D coordinates consistently with the method for the construction of Cer NP assemblies. Biopolymer module was then used to proceed with flexible torsions in the ligands and therefore all dihedral angles were allowed to rotate freely. The docking procedure was levied on the whole CER receptor without imposing the binding site (“blind docking”). A 60 × 60 × 60 grid box with a grid spacing of 0.375 A˚ was generated, which was large enough to totally cover the overall surface of the receptor. Affinity grid fields were obtained from the auxiliary program AutoGrid 3.0. Lamarckian genetic algorithm (LGA) was probed to find the most favorable complex geometry, i.e. the most favorable interactions. Optimized AutoDocking parameters were stated as follows: for LGA searching algorithms the maximum number of energy evaluations was increased to 25,000,000 per run; the iterations of Solis & Wets local search were 3000; the number of individuals in population was set to 300 and the number of generations was set to 100. All other parameters were set to the default settings. Cluster analysis of AutoDock results was carried out to determine whether different binding sites have generated from multiple runs (Morris et al., 1998). 2.7. TEWL experiments The rats were anesthetized with urethane (20%, w/v, i.p.) and kept under the ambient conditions (22 ± 2 ◦ C, RH 50 ± 5%) after removal of abdominal hair. The central abdomen of the rats was then divided into 6 different sections with each area of 1 cm2 . TEWL was recorded using an open-chamber Tewameter® (TM 300, Courage & Khazaka Co., Germany). Measurement was taken at a stable level 60 s after application of the TEWL probe to the skin. TEWL was automatically calculated and expressed in g m−2 h−1 . In order to minimize inter sample variation, an initial TEWL of the area before treatment was determined and recorded as TEWLuntreated for normalization. Then the six marked areas were respectively treated by 10 l each enhancer solution (10 mg MT or its analogues dissolved in 100 l ethanol) for an exposure time of 30 min, which was in accordance with the skin treatment procedure in ATR-FTIR experiments. At the end of the skin treatment, measurement was restarted and TEWLtreated was recorded. Each experiment was performed in quadruplicates. ERTEWL was calculated as follows: ERTEWL =
TEW Ltreated TEW Luntreated
(3)
2.8. Statistical analysis Results are expressed as mean ± S.E. The data were subjected to analysis of variance (ANOVA) using SPSS 16.0 software. A significant level was taken as p < 0.05.
Fig. 3. Cumulative penetrated amount (Q8 h ) of 5-FU in the presence of MT and its analogues.
3. Results and discussion 3.1. Structure–activity relationship for unsaturated MT analogues The rat skin can be used to gain the general insights into the transdermal pattern and acting mechanisms for penetration enhancers, though it is not a precise model for human skin for the percutaneous absorption (Bronaugh et al., 1982; Scott et al., 1992; Godin and Touitou, 2007). The promoting activity of the unsaturated MT analogues was assessed by comparison with the control group without addition of any enhancers and MT group. Q8 h of 5-FU penetrated across the rat skin was shown in Fig. 3. Compared with control, all the MT analogues exhibited enhancement activity (p < 0.05). Especially, Compd 3 which possessed an exocyclic double bond at C (4) showed a significantly better activity than MT, achieving an ERflux of 3.08 (p < 0.05). Other MT analogues were less effective than Compd 3 (p < 0.05), and no significant difference was found between them and MT (p > 0.05). The percutaneous permeation parameters (Q8 h , Js , Tlag , ERQ , ERflux ) and drug solubility in IPM vehicle were presented in Table 1. All the MT analogues and MT increased the drug solubility, however, no significant difference could be detected between Compd 3 and other analogues (p > 0.05). It was therefore reasonable to speculate that solubility enhancement was not the main factor determining the excellent activity of Compd 3. It was clear that not all the unsaturated MT analogues had better promoting activity than MT towards the transdermal penetration of 5-FU, in spite of their high structural similarity. The enhancing activity of these compounds was strongly associated with the molecular position of the double bond. Therefore, a mechanism study at a molecular level was suggested to understand the acting mechanisms for MT and its analogues. 3.2. ATR-FTIR experiments By ATR-FTIR, the CH2 asymmetric (as CH2 , 2920 cm−1 ) and symmetric (s CH2 , 2850 cm−1 ) stretching vibrations are commonly used as parameters for evaluating the molecular organization of the lipid alkyl chains. A blue shift in these peaks indicates that the vibration and disorder of the hydrocarbon chains might occur (Guillard et al., 2009; Obata et al., 2010). The present experiments of ATR-FTIR were carried out with MT and its best analogue, Compd 3. Ethanol was used as an ideal solvent mainly for two reasons. First, many studies have revealed that the ethanol alone is
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Table 1 Transdermal permeation parameters and drug solubility in the donor vehicle.
* #
Enhancers
Solubility (g ml−1 )
Control MT Compd 1 Compd 2 Compd 3 Compd 4
14.64 25.81 26.26 25.10 26.70 27.13
± ± ± ± ± ±
1.64 0.99* 1.76* 2.14* 1.27* 0.44*
Tlag (h)
Js (g cm−2 h−1 )
−0.32 −0.05 −0.27 −0.52 −0.23 0.08
3.20 4.43 4.47 4.96 9.84 5.80
± ± ± ± ± ±
0.08 0.81* 0.13* 0.53* 0.95*,# 1.79*
Q8 h (g cm−2 )
ERQ
ERflux
± ± ± ± ± ±
1 1.40 1.29 1.47 2.69 1.61
1 1.38 1.40 1.55 3.08 1.81
26.69 37.48 34.51 39.23 71.88 43.12
1.92 5.20* 0.49* 0.50* 7.45*,# 7.18*
Value is significantly superior to control group (p < 0.05). Value is significantly superior to the MT group (p < 0.05).
inefficient to produce any changes in the C H stretching absorbance from SC lipids (Bhatia et al., 1997; Zhao and Singh, 1998). Second, 10 l alcohol used in the experiments could be completely evaporated within one minute and a rapid reversibility of its action would be expected after application on the skin (Ibrahim and Li, 2009). Consequently, exposure of the rat skin to ethanol would not affect the determination of ATR-FTIR or TEWL. The results could simply reflect the effect of penetration enhancer on the molecular organization of the intercellular lipids in SC. Amphiphilic enhancers possessing a long alkyl chain are likely to be inserted into the hydrophobic chains of the SC lipids and to induce disturbance of the molecular organization of the hydrophobic domain (Vávrová et al., 2005). Therefore, an amphiphilic enhancer, 2-isopropyl-5methylcyclohexyl heptanoate (M-HEP) was used as a reference of alkyl chain disruptor. The result was illustrated in Fig. 4. Compared with control, no significant difference in the peak position of as CH2 or s CH2 could be observed after skin treatment by MT or Compd 3. None of them produced a blue or a red shift in these stretching peak positions. By contrast, M-HEP resulted in the shift of s CH2 and as CH2 significantly towards higher wavenumbers. Similar results were also obtained in the study of other terpene compounds, such as eugenol, limonene and menthone (Zhao and Singh, 1998; Krishnaiah et al., 2002). Obata et al. (2010) found that significant absorption shift (>1 cm−1 ) by menthol could only occur at 45 ◦ C. Based on these findings, it was likely that the studied compounds did not act by associating or interacting with the hydrophobic tails of the skin lipids, and the polar moieties might be the targeting site for the action of enhancement. The amide I (1650 cm−1 ) and amide II (1650 cm−1 ) in ATR-FTIR spectra bands were previously used to assess the breakage of H-bond net by the application of penetration enhancers (Jain et al., 2002). However, it has been clearly demonstrated that
these vibrations are derived from the amide bonds of the keratin in coreocytes, but not from the intercellular lipids (Obata et al., 2010). Consequently, these spectra bonds were not taken into consideration in our study. Instead, molecular modeling has been proposed to investigate the structural change in the polar head region of the skin lipids (Narishetty and Panchagnula, 2004).
Fig. 4. ATR-FTIR spectra of rat epidermis illustrating the presence of peaks near 2920 cm−1 and 2850 cm−1 after treatment by control, M-HEP, MT and Compd 3.
Fig. 5. Chemical structures of Cer NP (a) and the polymer chain assemblies of Cer NP (b).
3.3. Molecular modeling Ceramides in the lipids of SC are considered as the essential component for maintaining the skin barrier function (Bouwstra et al., 1999). Among them, ceramide NP (Cer NP, Fig. 5a), equivalent to human ceramide 3, was often used to investigate the effect of penetration enhancers on the skin lipids due to its important role in the lipid organization of SC barrier (Imura et al., 2001; Rerek et al., 2005). In the present study, one Cer NP was first selected as a repeated unit and then eight Cer NP molecules were optimized together to produce favorable polymer chain assemblies of Cer NP (Fig. 5b), which was used to simulate a microenvironment of the ceramides in SC. Then the drug or enhancer was added into the system separately and the conformation continued to be optimized until an appropriate one was produced, as shown in Fig. 6. As shown in Fig. 6a, 5-FU partitioned into the polar domain of the ceramides via H-bonds and van der waals forces, indicating that it had good affinity to the polar lipid domain in the SC. A similar associating site could also be observed for MT and its analogues, which was shown in Fig. 6b–f. All the compounds located at polar domain (red color containing area) and formed H-bonds with the polar headgroups of the Cer NP assemblies. Based on the optimal conformation, the
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Fig. 6. Interaction of the Cer NP assemblies with 5-FU (a) and different penetration enhancers, including MT (b), Compd 1 (c), Compd 2 (d), Compd 3 (e) and Compd 4 (f). These figures are screenshots of the polar moieties of the Cer NP assemblies. H-bonds were presented in brown dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
docked energy for 5-FU, MT, Compd 1, Compd 2, Compd 3 and Compd 4 was −4.33 kcal mol−1 , −2.79 kcal mol−1 , −4.20 kcal mol−1 , −3.86 kcal mol−1 , −5.03 kcal mol−1 , −4.21 kcal mol−1 , respectively. The values were generally negative, suggesting that the interaction of Cer NP assemblies with the drug or enhancers was possible. The intercellular spaces of SC contain structured lipids and a diffusing molecule has to cross a variety of hydrophobic and polar domains before it reaches the viable epidermis (Hadgraft, 2004). There was a possibility that the occupation of enhancers in the
polar lipid domain could disturb the interaction between the drug and ceramides, pulling the drug free of H-bonds and increasing the diffusivity across SC. Additionally, molecular interaction would be favorable when the docked energy was low. Among all these compounds, the lowest docked energy was provided by Compd 3, potentially indicative that it had the greatest affinity to the polar groups of the ceramides. It could preferentially interact with the polar group of the lipids, which offered a reasonable explanation for its best enhancement activity.
Fig. 7. (a) The enhancement ratio of the TEWL (ERTEWL ) through the rat skin in the presence of pure ethanol, MT and its analogues in the solution of ethanol. (b) The correlation between ERflux and ERTEWL , R2 = 0.92.
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3.4. TEWL experiments Water molecule was selected as a suitable probe to investigate the possible change of the polar lipid domain induced by enhancer treatment. It has been demonstrated that the orderly arranged intercellular lipids form an important barrier to TEWL (Elias, 1983; Downing et al., 1987). The polar head groups of intercellular lipids provided significant binding sites for the water in the skin (Charalambopoulou et al., 2004; Verdier-Sévrain and Bonté, 2007; Nakazawa et al., 2012). Water transport across the skin can be mediated by the modification of the lipid organization (VerdierSévrain and Bonté, 2007). Determination of the outward diffusion of water through the skin could reflect the structural alteration in the polar lipid domains. Therefore, TEWL was measured in the study to further investigate the change of transdermal polar pathway induced by various MT analogues. The initial TEWL of untreated skin fell into the range of 4.0–8.0 g m−2 h−1 , which agreed well with a previous report (Meguro et al., 2000). Skin treatment of different compounds was carried out, consistently with ATR-FTIR experiments. As shown in Fig. 7a, the control group caused little change in the transdermal polar domain in term of TEWL, but a significant increase was observed after the skin treatment of different enhancers. Compd 3 showed the highest ERTEWL , which was correlated well with the results of the in vitro penetration experiments and molecular modeling. As shown in Fig. 7b, a linear correlation (R2 = 0.92) was found between ERTEWL and ERflux , which was true for ERQ as well. Based on these findings, it was reasonable to speculate that these compounds modified the transdermal polar pathway by interacting with the polar headgroups of the skin lipid, and then increase the diffusion of hydrophilic compounds. The present study agreed well with the previous assumption of Cornwell and Barry (1993) and Narishetty and Panchagnula (2004), who suggested that polar pathway might play an important role in the terpene enhancement towards the transdermal absorption of the hydrophilic drugs. 4. Conclusion The study revealed that Compd 3 with an exocyclic double bond at C (4) had a significantly better enhancement activity than MT and other unsaturated analogues in the transdermal absorption of 5-FU. It was suggested that these compounds could modify the transdermal polar pathway, effectively by interacting with the polar domain of the intercellular lipids without affecting the conformational order of the hydrophobic chains. The attempts in this study represented some new ideas for the penetration enhancer design and extended the understanding about the acting mechanisms for enhancers. Acknowledgement This work was supported by National Natural Science Foundation of China (No: 81173007). References Aqil, M., Ahad, A., Sultana, Y., Ali, A., 2007. Status of terpenes as skin penetration enhancers. Drug Discov. Today 12, 1061–1067. Barry, B.W., 1991. Lipid–protein-partitioning theory of skin penetration enhancement. J. Control. Release 15, 237–248. Barry, B.W., 2001. Novel mechanisms and devices to enable successful transdermal drug delivery. Eur. J. Pharm. Sci. 14, 101–114. Bhatia, K.S., Gao, S., Singh, J., 1997. Effect of penetration enhancers and iontophoresis on the FT-IR spectroscopy and LHRH permeability through porcine skin. J. Control. Release 47, 81–89. Bouwstra, J.A., Dubbelaar, F.E.R., Gooris, G.S., Weerheim, A.M., Ponec, M., 1999. The role of ceramide composition in the lipid organization of the skin barrier. BBA – Biomembranes 1419, 127–136.
Bronaugh, R.L., Stewart, R.F., Congdon, E.R., 1982. Methods for in vitro percutaneous absorption studies II. Animal models for human skin. Toxicol. Appl. Pharmacol. 62, 481–488. Chantasart, D., Pongjanyakul, T., Higuchi, W.I., Li, S.K., 2009. Effects of oxygencontaining terpenes as skin permeation enhancers on the lipoidal pathways of human epidermal membrane. J. Pharm. Sci. 98, 3617–3632. Charalambopoulou, G.C., Steriotis, T.A., Hauss, T., Stubos, A.K., Kanellopoulos, N.K., 2004. Structural alterations of fully hydrated human stratum corneum. Physica B 350, E603–E606. Cornwell, P.A., Barry, B.W., 1993. The routes of penetration of ions and 5-fluorouracil across human skin and the mechanisms of action of terpene skin penetration enhancers. Int. J. Pharm. 21, 189–194. Downing, D.T., Stewart, M.E., Wertz, P.W., Colton, S.W., Abraham, W., Strauss, J.S., 1987. Skin lipids: an update. J. Invest. Dermatol. 88, S2–S6. Elias, P.M., 1983. Epidermal lipids, barrier function, and desquamation. J. Invest. Dermatol. 80, S44–S49. Fang, L., Kobayashi, Y., Numajiri, S., Kobayashi, D., Sugibayashi, K., Morimoto, Y., 2002. The enhancing effect of a triethanolamine-ethanol-isopropyl myristate mixed system on the skin permeation of acidic drugs. Biol. Pharm. Bull. 25, 1339–1344. Finnin, B.C., Morgan, T.M., 1999. Transdermal penetration enhancers: applications, limitations, and potential. J. Pharm. Sci. 88, 955–958. 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. Godin, B., Touitou, E., 2007. Transdermal skin delivery: Predictions for humans from in vivo, ex vivo and animal models. Adv. Drug. Deliv. Rev. 59, 1152–1161. Guillard, E.C., Tfayli, A., Laugel, C., Baillet-Guffroy, A., 2009. Molecular interactions of penetration enhancers within ceramides organization: a FTIR approach. Eur. J. Pharm. Sci. 36, 192–199. Hadgraft, J., 2004. Skin deep. Eur. J. Pharm. Biopharm. 58, 291–299. Ibrahim, S.A., Li, S.K., 2009. Effects of solvent deposited enhancers on transdermal permeation and their relationship with Emax . J. Control. Release 136, 117–124. Imura, T., Sakai, H., Yamauchi, H., Kaise, C., Kozawa, K., Yokoyama, S., Abe, M., 2001. Preparation of liposomes containing ceramide 3 and their membrane characteristics. Colloids Surf. B 20, 1–8. Iyer, M., Zheng, T., Hopfinger, A.J., Tseng, Y.J., 2007. QSAR analyses of skin penetration enhancers. J. Chem. Inf. Model. 47, 1130–1149. Jain, A.K., Thomas, N.S., Panchagnula, R., 2002. Transdermal drug delivery of imipramine hydrochloride. I. Effect of terpenes. J. Control. Release 79, 93–101. Jain, A., Karande, P., Mitragotri, S., 2006. High throughput screening of transdermal penetration enhancers: opportunities, methods, and applications. In: Smith, E.W., Maibach, H.I. (Eds.), Percutaneous Penetration Enhancers. CRC Press, Boca Raton, pp. 319–333. Kang, L., Yap, C.W., Lim, P.F.C., Chen, Y.Z., Ho, P.C., Chan, Y.W., Wong, G.P., Chan, S.Y., 2007. Formulation development of transdermal dosage forms: quantitative structure–activity relationship model for predicting activities of terpenes that enhance drug penetration through human skin. J. Control. Release 120, 211–219. Krishnaiah, Y.S.R., Satyanarayana, V., Karthikeyan, R.S., 2002. Penetration enhancing effect of menthol on the percutaneous flux of nicardipine hydrochloride through excised rat epidermis from hydroxypropyl cellulose gels. Pharm. Dev. Technol. 7, 305–315. Levin, J., Marbach, H., 2005. The correlation between transepidermal water loss and percutaneous absorption: an overview. J. Control. Release 103, 291–299. Meguro, S., Arai, Y., Masukawa, Y., Uie, K., Tokimitsu, I., 2000. Relationship between covalently bound ceramides and transepidermal water loss (TEWL). Arch. Dermatol. Res. 292, 463–468. Menon, G.K., 2002. New insights into skin structure: scratching the surface. Adv. Drug. Deliv. Rev. 54, S3–S17. Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., Olson, A.J., 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639–1662. Nakazawa, H., Ohta, N., Hatta, I., 2012. A possible regulation mechanism of water content in human stratum corneum via intercellular lipid matrix. Chem. Phys. Lipids 165, 238–243. Narishetty, S.T.K., Panchagnula, R., 2004. Transdermal delivery of zidovudine: effect of terpenes and their mechanism of action. J. Control. Release 95, 367–379. Obata, Y., Utsumi, S., Watanabe, H., Suda, M., Tokudome, Y., Otsuka, M., Takayama, K., 2010. Infrared spectroscopic study of lipid interaction in stratum corneum treated with transdermal absorption enhancers. Int. J. Pharm. 389, 18–23. Paquette, L.A., Doehner, R.F., 1980. Synthesis of optically active triazolinediones and examination of their utility for inducing asymmetry in Diels-Alder cycloaddition reactions. J. Org. Chem. 45, 5105–5113. Rerek, M.E., Van Wyck, D., Mendelsohn, R., Moore, D.J., 2005. FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix. Chem. Phys. Lipids. 134, 51–58. Serra, S., Brenna, E., Fuganti, C., Maggioni, F., 2003. Lipase-catalyzed resolution of pmenthan-3-ols monoterpenes: preparation of the enantiomer-enriched forms of menthol, isopulegol, trans- and cis-piperitol, and cis-isopiperitenol. Tetrahedron: Asymmetry 14, 3313–3319. Scott, R.C., Batten, P.L., Clowes, H.M., Jones, B.K., Ramsey, J.D., 1992. Further validation of an in vitro method to reduce the need for in vivo studies for measuring the absorption of chemicals through rat skin. Fundam. Appl. Toxicol. 19, 484–492. United States Pharmacopeia, 2009. Fluorouracil. In: USP 32-NF 27. 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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Effect of unsaturated menthol analogues on the in vitro penetration of 5-fluorouracil through rat skin Yang Chen a , Jian Wang a , Dongmei Cun a , Manli Wang a,b , Juan Jiang a , Honglei Xi a , Hongxia Cui a , Yongnan Xu a , Maosheng Cheng a , Liang Fang a,∗ a b
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, People’s Republic of China School of Pharmacy, Beihua University, 3999 Huashan Road, Jilin, Jilin 132013, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 16 October 2012 Accepted 7 January 2013 Available online 16 January 2013 Keywords: Transdermal Absorption enhancer Unsaturated Menthol analogue Mechanism Structure–activity relationship
a b s t r a c t To explore the structure–activity relationship for terpenes as transdermal penetration enhancers, unsaturated menthol analogues were synthesized in our study, including p-menth-1-en-3-ol (Compd 1), p-menth-4-en-3-ol (Compd 2), p-menth-4(8)-en-3-ol (Compd 3) and p-menth-8-en-3-ol (Compd 4). Their enhancing activity on the penetration of 5-fluorouracil through rat skin was evaluated by in vitro experiments. Attenuated total reflection-Fourier transform infrared spectroscopy, molecular modeling and transepidermal water loss (TEWL) were introduced to investigate the enhancer induced alteration in different skin lipid domains. The results indicated that Compd 3 achieved the highest enhancement ability with an enhancement ratio of 3.08. Other analogues were less effective than Compd 3, and no significant difference was found between them and menthol. Treatment of rat skin with these enhancers did not produce any shift in the stretching vibration of the methylene in hydrophobic lipid chains, but significantly improved the polar pathway across the rat skin as suggested by the increased TEWL. Molecular modeling results suggested that polar head groups of the skin lipids provided the main binding site for enhancer action. These findings indicated that the studied compounds enhanced drug transport by interacting with the polar domain of the skin lipid, instead of by affecting the arrangement of the hydrophobic chains. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The outermost layer of the skin, stratum corneum (SC), provides the principal rate limitation to the diffusion of drugs and water across the skin (Hadgraft, 2004). It is generally accepted that this barrier function is strongly associated with the complex and orderly organized structure of the intercellular lipids in SC (Wertz and van den Bergh, 1998; Menon, 2002). To enhance the skin permeability, many approaches have been used, including the chemical and physical techniques (Barry, 2001). The chemical approach has led to the introduction of transdermal penetration enhancers, which can be used to facilitate the percutaneous absorption by temporarily diminishing the impermeability of the skin (Finnin and Morgan, 1999; Jain et al., 2006). Terpenes represent one of the promising candidates for clinically acceptable enhancers (Aqil et al., 2007). Depending on the presence of carbon-carbon double bond, they can be classified as two types, i.e., saturated and unsaturated terpenes. Quantitative
∗ Corresponding author. Tel.: +86 24 23986330; fax: +86 24 23986330. E-mail address: [email protected] (L. Fang). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.015
structure–activity relationship (QSAR) study has revealed that the activity of terpenes is primarily related to their chemical structures (Ghafourian et al., 2004; Iyer et al., 2007; Kang et al., 2007), and the unsaturated terpenes seem to be better candidates than the saturated ones in promoting the transdermal absorption of 5fluorouracil (5-FU) (Kang et al., 2007). However, the inter laboratory difference and the large structural diversity of terpenes in the QSAR models might lead to a misjudgment of the result. Therefore, a detailed structural analysis about the terpenes is still needed to describe a precise structure–activity relationship. The present study was carried out to investigate the exact effect of double bond on the enhancement ability of the unsaturated terpenes. Menthol (MT), a saturated terpene that has widely been used as a penetration enhancer, was selected as the lead compound. A series of unsaturated analogues were obtained by introduction of a double bond onto its four different molecular positions, which gave the product of p-menth-1-en-3-ol (Compd 1), p-menth-4-en-3-ol (Compd 2), p-menth-4(8)-en-3-ol (Compd 3) and p-menth-8-en3-ol (Compd 4). Their enhancement effect on the transdermal permeability of a hydrophilic drug, 5-FU, was studied by comparison with MT. The structures of the drug, MT and its analogues were illustrated in Fig. 1. The strong structural similarity between MT
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Fig. 1. Chemical structures and molecular models of 5-FU, MT and its analogues. Carbon atoms were colored gray, oxygen atoms red, nitro blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and its unsaturated analogues made it possible to explore whether the double bond in unsaturated compounds could influence their transdermal promoting effect. One the other hand, although many studies have investigated the transdermal enhancement effects of terpenes, their exact mechanisms for action are not clearly defined (Chantasart et al., 2009). Data about the molecular interaction between SC lipids and enhancers, which plays an important role in understanding the mechanisms of enhancement, is still lacking so far. Previous studies have proposed that the effect of a penetration enhancer on the lipid bilayer may involve interactions at two sites, namely the polar head groups and the hydrophobic tails of the intercellular lipid (Barry, 1991). Therefore, in the present study, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was introduced to study the local effect of enhancers on the hydrophobic tails of the intercellular lipids (Guillard et al., 2009). Molecular modeling was used to observe the molecular interaction between enhancers and the polar head groups in SC lipids (Narishetty and Panchagnula, 2004). Finally, transepidermal water loss (TEWL) was selected as a noninvasive bioengineering method to investigate the possible alteration in polar pathway after skin treatment with different enhancers (Levin and Marbach, 2005). The objectives of the present study were to (a) determine the structure–enhancement relationship for MT and its unsaturated analogues, (b) respectively investigate the alterations in the
(1) Compd 1
hydrophobic moieties and polar domain of SC lipid after skin treatment with different enhancers, and (c) correlate these structural or functional changes with the enhancement activity towards 5-FU. 2. Materials and methods 2.1. Materials Compd 1, Compd 2 and Compd 3 were synthesized in our laboratory. Compd 4, piperitone and menthone were bought from TCI (Tokyo, Japan). Pulegone was obtained from Alfa Aesar (Ward Hill, MA, USA). 2-Isopropyl-5-methylcyclohexyl heptanoate (M-HEP) was prepared as described previously (Zhao et al., 2008). Isopropyl myristate (IPM) and MT were both purchased from China National Medicines Co., Ltd. (Shanghai, China). 5-FU was provided by Fangge Pharmaceutical Co., Ltd. (Zhejiang, China). Methanol of HPLC grade was supplied by the Yuwang Pharmaceutical Co., Ltd. (Shandong, China). All of other chemical reagents were of at least reagent grade available. 2.2. Synthesis of unsaturated MT analogues As shown in Fig. 2a and b, Compd 1 and Compd 3 were respectively prepared from piperitone and pulegone by LiAlH4 selective reduction (Serra et al., 2003). Briefly, a solution of corresponding substrate (5 g, 32.85 mmol) in absolute diethyl ether (15 ml) was
(2) Compd 2 a
a O
OH
O
OH
(3) Compd 3 b O
c Br
O
a O
OH
Fig. 2. Synthetic methods of Compd 1 (1), Compd 3 (2), and Compd 2 (3). a: LiAlH4 /Et2 O; b: N-bromosuccinide/benzoyl peroxide/CCl4 ; c: quinoline.
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added dropwise under nitrogen into a stirred equimolar suspension of LiAlH4 in absolute diethyl ester (50 ml) at 0 ◦ C. The temperature of the reaction was also kept at 0 ◦ C. After agitation for 2.5 h, the reaction was terminated by dropwise addition of water and 10% aqueous solution of NaOH. Then, the ether layer was separated, washed with brine, dried with anhydrous Na2 SO4 , and freed of volatile solvent under vacuum. The residue was purified by silica gel column chromatography with gradient elution consisting of ethyl acetate and petroleum ether (1:100→20:80) to give pure Compd 1 (yield, 82%), 1 H NMR (300 MHz, DMSO, ı): 0.752 and 0.894 (dd, J = 6.9 Hz, 6H, –CH(CH3 )2 ), 1.120–1.149 (m, 2H, –CH2 ), 1.364–1,384 (m, 1H, –CH), 1.602 (s, 3H, –CH3 ), 1.851–1.964 (m, 2H, –CH2 ), 1.964–2.057 (m, 1H, –CH), 3.774 (br, s, 1H, OH), 4.414 (d, J = 6.3 Hz, 1H, –O CH), 5.289 (s, 1H, CH); ESI-MS m/z: 154 (M+ ) and Compd 3 (yield, 78%), 1 H NMR (300 MHz, DMSO, ı): 0.912 (d, J = 6.3 Hz, 3H, –CH3 ), 1.155 (s, 6H, C(CH3 )2 ), 1.514–1.719 (m, 4H, –CH2 , –CH2 ), 1.972–2.130(m, 3H, –CH2 , –CH), 4.280 (s, 1H, –O CH), 5.582 (br, s, 1H, –OH); ESI-MS m/z: 154 (M+ ). The purity of Compd 1 and Compd 3 was over 95% as characterized by GC/MS. The synthetic sequence for Compd 2 was outlined in Fig. 2c. N-Bromosuccinide (8.4 g, 47.20 mmol) and benzoyl peroxide (1 g) were added to menthone (6 g, 32.91 mmol) which was dissolved in carbon tetrachloride (50 ml). The reaction mixture was then heated to reflux for 2 h while stirring. After being cooled and filtered, the obtained insoluble was washed with carbon tetrachloride, and the collected filtrates were washed successively with water, 10% sodium carbonate solution, water and brine. The solvent was removed under vacuum, and the residue was taken up in quinoline (11 g) and heated at 150 ◦ C for 30 min. The corresponding ketone product was afforded after a simple purification by chromatography eluting with ethyl acetate and petroleum ether (1:200→20:80) (Paquette and Doehner, 1980). The following step was performed in accordance with the synthetic scheme of Compd 1 and Compd 3, giving pure product of Compd 2 (yield, 50%), 1 H NMR (300 MHz, DMSO, ı): 0.902 (d, J = 7.5 Hz, 3H, –CHCH ), 3 0.937–0.983 (t, J = 6.9 Hz, 6H, –CH(CH3 )2 ), 1.135–1.174 (m, 1H, –CH), 1.557–1.606 (m, 2H, –CH2 ), 1.853–1.983 (m, 2H, –CH2 ), 2.502–2.623 (m, 1H, –CH), 4.519 (d, J = 6.6 Hz, 1H, –OCH), 5.290 (d, J = 4.2 Hz, 1H, CH); ESI-MS m/z: 154 (M+ ). The purity of Compd 2 was over 95% as characterized by GC/MS.
the guidelines for animal use published by the Life Science Research Center of Shenyang Pharmaceutical University. 2.3.3. In vitro permeation experiments Skin permeation experiments were performed according to the method of Fang et al. (2002). In vitro skin penetration experiments were carried out at 32 ◦ C in modified two-chamber diffusion cells. Each half-cell had a volume of 3 ml and an available diffusion area of 0.95 cm2 . The skin fragments were mounted onto the cells with the SC in contact with the donor chamber. The acceptor chamber was filled with 3 ml distilled water, and the donor chamber with drug suspension. Both compartments were continuously stirred with star-head bars at 600 rpm throughout the experiments. Samples (2 ml) from the acceptor chambers were withdrawn at predetermined intervals over 8 h, and analyzed by HPLC with the calibration of a standard curve. At each time point, the withdrawn sample was replaced with an equivalent volume of acceptor liquid. The experiments were performed in quadruplicates. 2.3.4. HPLC analysis of the drug The drug content was analyzed with an HPLC system consisting of an L-2420 variable-wavelength ultraviolet absorbance detector and an L-2130 pump (Hitachi High-Technologies Corporation, Tokyo, Japan). The reversed phase stainless-steel column (20 cm × 4.6 mm) was packed with Diamonsil C-18 (5 m particle size; Dikma Technologies, Beijing, China). The column temperature was maintained at 40 ◦ C. The eluent was detected at 265 nm. The mobile phase was composed entirely of distilled water and the flow rate was 1 ml min−1 (United States Pharmacopeia, 2009). 2.3.5. Data analysis The cumulative amount of penetrated drugs per unit area across the skin in 8 h (Q8 h , g cm−2 ) was calculated according to the measured drug concentration in receptor-phase after being corrected for sampling effects. The steady state flux (Js , g cm−2 h−1 ) and lag time (Tlag , h) were obtained from the slope and the x-intercept of the linear portion of the profile plotted between Q8 h on Y-axis and time on the X-axis. In order to normalize the promoting effect of different penetration enhancers, enhancement ratio (ERQ and ERflux ) was defined as follows: Q8 h (with enhancer) Q8 h (without enhancer)
(1)
Js (with enhancer) Js (without enhancer)
(2)
2.3. In vitro skin penetration experiments
ERQ =
2.3.1. Preparation of donor solutions Donor solutions were prepared by dissolving 5% (w/w) enhancers and excess amounts of 5-FU in IPM. Then the suspensions were stirred for 5 min, followed by sonication for 10 min and equilibration overnight. Control sample was obtained in a similar way without addition of any enhancers.
ERflux =
2.3.2. Skin preparation Full-thickness skins were gained from Male Wistar rats (180–220 g, 6–8 weeks old), which were provided by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). After the rats were anesthetized with urethane (20%, w/v, i.p.), the abdominal hair was removed by an electric clipper, and then carefully shaved with a razor. Full-thickness skins (i.e. epidermis with SC and dermis) were excised from the shaved abdomen sites of the sacrificed rats. The adhering subcutaneous fat and other extraneous tissue were surgically trimmed. After being washed and examined for integrity under the microscope, the skin was wrapped in aluminium foil and then subsequently stored at −70 ◦ C for a maximum of 2 weeks. The skins were allowed to thaw slowly prior to use in order to avoid damage. All the animal procedures were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and also in accordance with
2.4. Determination of drug solubility in donor solution An excessive amount of drug was added to the donor solution with or without enhancers. The mixture was shaken vigorously, and then allowed to equilibrate at 32 ± 1 ◦ C in a shaken water-bath for 48 h. Finally the suspension was filtered through a 0.45-m Millipore filter and analyzed by HPLC after appropriate dilution. The experiments were performed in quadruplicates. 2.5. ATR-FTIR experiment The rats were anesthetized with urethane (20%, w/v, i.p.) and the abdominal hair was shaved as mentioned above. Then the rat abdomen was divided into 4 different sections with each area of 1 cm2 . Ten microliter each enhancer solution (10 mg dissolved in 100 l ethanol) was applied onto the marked areas for an exposure time of 30 min. Skin treated with solvent alone was determined as control. After treatment, the skin was blotted with tissue paper and dried at room temperature for 15 min. Each area of diffusion was then subjected to ATR-FTIR analysis (NEXUS+70, Thermo Electron
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Corporation, MA, USA). ATR-FTIR spectra were recorded at 2 cm−1 resolution. 2.6. Molecular modeling Eight-times repeat units of ceramide NP (Cer NP) were built in three-dimensional (3D) coordinates by means of Sybyl 6.91 software package (Tripos, Inc., St. Louis, MO, USA). After addition of all the hydrogen atoms with Amber 7 FF99 force field, partial charges were assigned to Cer NP molecules according to the method of Gastei-ger-Hückel. The structures were subjected to further energy minimization using both steepest descent and conjugate gradient protocols. Docking calculations were preformed on AutoDock 3.05 (the Scripps Research Institute, La Jolla, FL, USA). The fully flexible 5FU, MT and its analogues, were built in 3D coordinates consistently with the method for the construction of Cer NP assemblies. Biopolymer module was then used to proceed with flexible torsions in the ligands and therefore all dihedral angles were allowed to rotate freely. The docking procedure was levied on the whole CER receptor without imposing the binding site (“blind docking”). A 60 × 60 × 60 grid box with a grid spacing of 0.375 A˚ was generated, which was large enough to totally cover the overall surface of the receptor. Affinity grid fields were obtained from the auxiliary program AutoGrid 3.0. Lamarckian genetic algorithm (LGA) was probed to find the most favorable complex geometry, i.e. the most favorable interactions. Optimized AutoDocking parameters were stated as follows: for LGA searching algorithms the maximum number of energy evaluations was increased to 25,000,000 per run; the iterations of Solis & Wets local search were 3000; the number of individuals in population was set to 300 and the number of generations was set to 100. All other parameters were set to the default settings. Cluster analysis of AutoDock results was carried out to determine whether different binding sites have generated from multiple runs (Morris et al., 1998). 2.7. TEWL experiments The rats were anesthetized with urethane (20%, w/v, i.p.) and kept under the ambient conditions (22 ± 2 ◦ C, RH 50 ± 5%) after removal of abdominal hair. The central abdomen of the rats was then divided into 6 different sections with each area of 1 cm2 . TEWL was recorded using an open-chamber Tewameter® (TM 300, Courage & Khazaka Co., Germany). Measurement was taken at a stable level 60 s after application of the TEWL probe to the skin. TEWL was automatically calculated and expressed in g m−2 h−1 . In order to minimize inter sample variation, an initial TEWL of the area before treatment was determined and recorded as TEWLuntreated for normalization. Then the six marked areas were respectively treated by 10 l each enhancer solution (10 mg MT or its analogues dissolved in 100 l ethanol) for an exposure time of 30 min, which was in accordance with the skin treatment procedure in ATR-FTIR experiments. At the end of the skin treatment, measurement was restarted and TEWLtreated was recorded. Each experiment was performed in quadruplicates. ERTEWL was calculated as follows: ERTEWL =
TEW Ltreated TEW Luntreated
(3)
2.8. Statistical analysis Results are expressed as mean ± S.E. The data were subjected to analysis of variance (ANOVA) using SPSS 16.0 software. A significant level was taken as p < 0.05.
Fig. 3. Cumulative penetrated amount (Q8 h ) of 5-FU in the presence of MT and its analogues.
3. Results and discussion 3.1. Structure–activity relationship for unsaturated MT analogues The rat skin can be used to gain the general insights into the transdermal pattern and acting mechanisms for penetration enhancers, though it is not a precise model for human skin for the percutaneous absorption (Bronaugh et al., 1982; Scott et al., 1992; Godin and Touitou, 2007). The promoting activity of the unsaturated MT analogues was assessed by comparison with the control group without addition of any enhancers and MT group. Q8 h of 5-FU penetrated across the rat skin was shown in Fig. 3. Compared with control, all the MT analogues exhibited enhancement activity (p < 0.05). Especially, Compd 3 which possessed an exocyclic double bond at C (4) showed a significantly better activity than MT, achieving an ERflux of 3.08 (p < 0.05). Other MT analogues were less effective than Compd 3 (p < 0.05), and no significant difference was found between them and MT (p > 0.05). The percutaneous permeation parameters (Q8 h , Js , Tlag , ERQ , ERflux ) and drug solubility in IPM vehicle were presented in Table 1. All the MT analogues and MT increased the drug solubility, however, no significant difference could be detected between Compd 3 and other analogues (p > 0.05). It was therefore reasonable to speculate that solubility enhancement was not the main factor determining the excellent activity of Compd 3. It was clear that not all the unsaturated MT analogues had better promoting activity than MT towards the transdermal penetration of 5-FU, in spite of their high structural similarity. The enhancing activity of these compounds was strongly associated with the molecular position of the double bond. Therefore, a mechanism study at a molecular level was suggested to understand the acting mechanisms for MT and its analogues. 3.2. ATR-FTIR experiments By ATR-FTIR, the CH2 asymmetric (as CH2 , 2920 cm−1 ) and symmetric (s CH2 , 2850 cm−1 ) stretching vibrations are commonly used as parameters for evaluating the molecular organization of the lipid alkyl chains. A blue shift in these peaks indicates that the vibration and disorder of the hydrocarbon chains might occur (Guillard et al., 2009; Obata et al., 2010). The present experiments of ATR-FTIR were carried out with MT and its best analogue, Compd 3. Ethanol was used as an ideal solvent mainly for two reasons. First, many studies have revealed that the ethanol alone is
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Table 1 Transdermal permeation parameters and drug solubility in the donor vehicle.
* #
Enhancers
Solubility (g ml−1 )
Control MT Compd 1 Compd 2 Compd 3 Compd 4
14.64 25.81 26.26 25.10 26.70 27.13
± ± ± ± ± ±
1.64 0.99* 1.76* 2.14* 1.27* 0.44*
Tlag (h)
Js (g cm−2 h−1 )
−0.32 −0.05 −0.27 −0.52 −0.23 0.08
3.20 4.43 4.47 4.96 9.84 5.80
± ± ± ± ± ±
0.08 0.81* 0.13* 0.53* 0.95*,# 1.79*
Q8 h (g cm−2 )
ERQ
ERflux
± ± ± ± ± ±
1 1.40 1.29 1.47 2.69 1.61
1 1.38 1.40 1.55 3.08 1.81
26.69 37.48 34.51 39.23 71.88 43.12
1.92 5.20* 0.49* 0.50* 7.45*,# 7.18*
Value is significantly superior to control group (p < 0.05). Value is significantly superior to the MT group (p < 0.05).
inefficient to produce any changes in the C H stretching absorbance from SC lipids (Bhatia et al., 1997; Zhao and Singh, 1998). Second, 10 l alcohol used in the experiments could be completely evaporated within one minute and a rapid reversibility of its action would be expected after application on the skin (Ibrahim and Li, 2009). Consequently, exposure of the rat skin to ethanol would not affect the determination of ATR-FTIR or TEWL. The results could simply reflect the effect of penetration enhancer on the molecular organization of the intercellular lipids in SC. Amphiphilic enhancers possessing a long alkyl chain are likely to be inserted into the hydrophobic chains of the SC lipids and to induce disturbance of the molecular organization of the hydrophobic domain (Vávrová et al., 2005). Therefore, an amphiphilic enhancer, 2-isopropyl-5methylcyclohexyl heptanoate (M-HEP) was used as a reference of alkyl chain disruptor. The result was illustrated in Fig. 4. Compared with control, no significant difference in the peak position of as CH2 or s CH2 could be observed after skin treatment by MT or Compd 3. None of them produced a blue or a red shift in these stretching peak positions. By contrast, M-HEP resulted in the shift of s CH2 and as CH2 significantly towards higher wavenumbers. Similar results were also obtained in the study of other terpene compounds, such as eugenol, limonene and menthone (Zhao and Singh, 1998; Krishnaiah et al., 2002). Obata et al. (2010) found that significant absorption shift (>1 cm−1 ) by menthol could only occur at 45 ◦ C. Based on these findings, it was likely that the studied compounds did not act by associating or interacting with the hydrophobic tails of the skin lipids, and the polar moieties might be the targeting site for the action of enhancement. The amide I (1650 cm−1 ) and amide II (1650 cm−1 ) in ATR-FTIR spectra bands were previously used to assess the breakage of H-bond net by the application of penetration enhancers (Jain et al., 2002). However, it has been clearly demonstrated that
these vibrations are derived from the amide bonds of the keratin in coreocytes, but not from the intercellular lipids (Obata et al., 2010). Consequently, these spectra bonds were not taken into consideration in our study. Instead, molecular modeling has been proposed to investigate the structural change in the polar head region of the skin lipids (Narishetty and Panchagnula, 2004).
Fig. 4. ATR-FTIR spectra of rat epidermis illustrating the presence of peaks near 2920 cm−1 and 2850 cm−1 after treatment by control, M-HEP, MT and Compd 3.
Fig. 5. Chemical structures of Cer NP (a) and the polymer chain assemblies of Cer NP (b).
3.3. Molecular modeling Ceramides in the lipids of SC are considered as the essential component for maintaining the skin barrier function (Bouwstra et al., 1999). Among them, ceramide NP (Cer NP, Fig. 5a), equivalent to human ceramide 3, was often used to investigate the effect of penetration enhancers on the skin lipids due to its important role in the lipid organization of SC barrier (Imura et al., 2001; Rerek et al., 2005). In the present study, one Cer NP was first selected as a repeated unit and then eight Cer NP molecules were optimized together to produce favorable polymer chain assemblies of Cer NP (Fig. 5b), which was used to simulate a microenvironment of the ceramides in SC. Then the drug or enhancer was added into the system separately and the conformation continued to be optimized until an appropriate one was produced, as shown in Fig. 6. As shown in Fig. 6a, 5-FU partitioned into the polar domain of the ceramides via H-bonds and van der waals forces, indicating that it had good affinity to the polar lipid domain in the SC. A similar associating site could also be observed for MT and its analogues, which was shown in Fig. 6b–f. All the compounds located at polar domain (red color containing area) and formed H-bonds with the polar headgroups of the Cer NP assemblies. Based on the optimal conformation, the
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Fig. 6. Interaction of the Cer NP assemblies with 5-FU (a) and different penetration enhancers, including MT (b), Compd 1 (c), Compd 2 (d), Compd 3 (e) and Compd 4 (f). These figures are screenshots of the polar moieties of the Cer NP assemblies. H-bonds were presented in brown dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
docked energy for 5-FU, MT, Compd 1, Compd 2, Compd 3 and Compd 4 was −4.33 kcal mol−1 , −2.79 kcal mol−1 , −4.20 kcal mol−1 , −3.86 kcal mol−1 , −5.03 kcal mol−1 , −4.21 kcal mol−1 , respectively. The values were generally negative, suggesting that the interaction of Cer NP assemblies with the drug or enhancers was possible. The intercellular spaces of SC contain structured lipids and a diffusing molecule has to cross a variety of hydrophobic and polar domains before it reaches the viable epidermis (Hadgraft, 2004). There was a possibility that the occupation of enhancers in the
polar lipid domain could disturb the interaction between the drug and ceramides, pulling the drug free of H-bonds and increasing the diffusivity across SC. Additionally, molecular interaction would be favorable when the docked energy was low. Among all these compounds, the lowest docked energy was provided by Compd 3, potentially indicative that it had the greatest affinity to the polar groups of the ceramides. It could preferentially interact with the polar group of the lipids, which offered a reasonable explanation for its best enhancement activity.
Fig. 7. (a) The enhancement ratio of the TEWL (ERTEWL ) through the rat skin in the presence of pure ethanol, MT and its analogues in the solution of ethanol. (b) The correlation between ERflux and ERTEWL , R2 = 0.92.
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3.4. TEWL experiments Water molecule was selected as a suitable probe to investigate the possible change of the polar lipid domain induced by enhancer treatment. It has been demonstrated that the orderly arranged intercellular lipids form an important barrier to TEWL (Elias, 1983; Downing et al., 1987). The polar head groups of intercellular lipids provided significant binding sites for the water in the skin (Charalambopoulou et al., 2004; Verdier-Sévrain and Bonté, 2007; Nakazawa et al., 2012). Water transport across the skin can be mediated by the modification of the lipid organization (VerdierSévrain and Bonté, 2007). Determination of the outward diffusion of water through the skin could reflect the structural alteration in the polar lipid domains. Therefore, TEWL was measured in the study to further investigate the change of transdermal polar pathway induced by various MT analogues. The initial TEWL of untreated skin fell into the range of 4.0–8.0 g m−2 h−1 , which agreed well with a previous report (Meguro et al., 2000). Skin treatment of different compounds was carried out, consistently with ATR-FTIR experiments. As shown in Fig. 7a, the control group caused little change in the transdermal polar domain in term of TEWL, but a significant increase was observed after the skin treatment of different enhancers. Compd 3 showed the highest ERTEWL , which was correlated well with the results of the in vitro penetration experiments and molecular modeling. As shown in Fig. 7b, a linear correlation (R2 = 0.92) was found between ERTEWL and ERflux , which was true for ERQ as well. Based on these findings, it was reasonable to speculate that these compounds modified the transdermal polar pathway by interacting with the polar headgroups of the skin lipid, and then increase the diffusion of hydrophilic compounds. The present study agreed well with the previous assumption of Cornwell and Barry (1993) and Narishetty and Panchagnula (2004), who suggested that polar pathway might play an important role in the terpene enhancement towards the transdermal absorption of the hydrophilic drugs. 4. Conclusion The study revealed that Compd 3 with an exocyclic double bond at C (4) had a significantly better enhancement activity than MT and other unsaturated analogues in the transdermal absorption of 5-FU. It was suggested that these compounds could modify the transdermal polar pathway, effectively by interacting with the polar domain of the intercellular lipids without affecting the conformational order of the hydrophobic chains. The attempts in this study represented some new ideas for the penetration enhancer design and extended the understanding about the acting mechanisms for enhancers. Acknowledgement This work was supported by National Natural Science Foundation of China (No: 81173007). References Aqil, M., Ahad, A., Sultana, Y., Ali, A., 2007. Status of terpenes as skin penetration enhancers. Drug Discov. Today 12, 1061–1067. Barry, B.W., 1991. Lipid–protein-partitioning theory of skin penetration enhancement. J. Control. Release 15, 237–248. Barry, B.W., 2001. Novel mechanisms and devices to enable successful transdermal drug delivery. Eur. J. Pharm. Sci. 14, 101–114. Bhatia, K.S., Gao, S., Singh, J., 1997. Effect of penetration enhancers and iontophoresis on the FT-IR spectroscopy and LHRH permeability through porcine skin. J. Control. Release 47, 81–89. Bouwstra, J.A., Dubbelaar, F.E.R., Gooris, G.S., Weerheim, A.M., Ponec, M., 1999. The role of ceramide composition in the lipid organization of the skin barrier. BBA – Biomembranes 1419, 127–136.
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