Drug Delivery
ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20
Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin Meng Yu, Fang Guo, Ying Ling, Nan Li & Fengping Tan To cite this article: Meng Yu, Fang Guo, Ying Ling, Nan Li & Fengping Tan (2013): Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin, Drug Delivery To link to this article: http://dx.doi.org/10.3109/10717544.2013.869276
Published online: 13 Dec 2013.
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Date: 05 November 2015, At: 21:56
http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Delivery, Early Online: 1–6 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.869276
Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin Meng Yu, Fang Guo, Ying Ling, Nan Li, and Fengping Tan
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Tianjin Key Laboratory of Drug Delivery and High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, PR China
Abstract
Keywords
Objective: This study was to investigate the topical skin targeting effects and mechanism of combination penetration modifiers of 1,2-hexanediol (or 1,2-heptanediol) and 1,4-cyclohexanediol on transdermal absorption of metronidazole (MTZ) in different skin models. Methods: Six formulations were applied to pig abdominal skin and pig ear skin models, respectively, and the results were compared with the previous data on hairless mouse skin worked out by our laboratory. Four parameters (flux, Tlag, Q24 and targeting ratio) were used to evaluate permeability and targeting effect in skin. Results: The combined penetration modifiers played a general role on decreasing permeability without reducing skin retention. The most significant skin permeability decrement to MTZ was pig abdominal skin (permeability decrement was 20% for hairless mouse skin, 60% for pig abdominal skin and 40% for pig ear skin, respectively) while the strongest skin targeting effect appeared in hairless mouse skin (targeting ratios were 1.79 for hairless mouse skin, 1.24 for pig abdominal skin and 1.05 for pig ear skin, respectively) under the role of penetration modifiers. Conclusions: Thickness of stratum corneum (SC) was the major factor impact on skin targeting effect. Selection criteria of skin models also have been discussed in this study.
Animal skin model, penetration modifier, skin targeting, transdermal absorption, transfer pathway
Introduction Transdermal drug delivery is a non-invasive and user-friendly delivery method. Traditionally, transdermal formulation was designed to achieve systemic therapeutics by transfering drug into blood circulation through skin layers. Physical and chemical methods have been developed to overcome the remarkable barrier properties of the outermost layer of skin, the stratum corneum (SC) and enhance the transdermal delivery of drugs (Doh et al., 2003; Qiu et al., 2008; Rossetti et al., 2011). However, transdermal formulation attracted much attention on topical skin targeting application in recent decades. Active agents from applied formulations were able to penetrate the SC layer and reach dermatologically viable layer to play a therapeutic role. However they were also able to penetrate into systematic circulation and lead to toxic side effects. In this study, a nitroimidazole derivative metronidazole (MTZ) was used as a model drug, which was expected to skin targeting for therapy of acne and rosacea. MTZ was an acidic drug with pKa 2.6, it was able to penetrate the skin effectively for its small molecular weight (Mw ¼ 171.15 g/mol), but it did not indicating potential retention within lipid domains and the establishment of a reservoir for its low lipophicity (logP ¼ 0.18). For treatment of dermatological conditions, an ideal topical formulation is to impart maximal skin Address for correspondence: Nan Li, Tianjin Key Laboratory of Drug Delivery and High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072 Tianjin, PR China. Tel: þ86 022 27404986. Email: [email protected]
History Received 16 August 2013 Accepted 21 November 2013
retention and minimal systematic penetration. Thus, there is a significant need in discovering safe and effective skin targeting measures. Varying measures have been published to enhance skin retention, such as making prodrugs (Hsieh et al., 2012), using vehicles (¸Senyig˘it et al., 2010; Tsai et al., 2010) and adding penetration modifiers. Adding penetration modifiers has significant advantages over other measures for ease of manipulation, available without specific devices. A plenty of researches have been done to improve penetration modifiers. Hadgraft et al. (1996) have reported that some Azone derivative might inhibit penetration of MTZ efficiently. Asbill & Michniak (2000) successfully prevented drug from transfering into deeper skin layers by using chemical skin modifiers. In the previous study of our laboratory, skin targeting effects of 1,4-cyclohexanediol alone and synergistic with 1,2-hexanediol have been validated (Li et al., 2010, 2011). Although the effect of penetration modifiers have been investigated in plenty of researches, various animal skin models were used depended on different purposes. There was not a specifically animal skin model as a surrogate for human in in vitro penetration studies. Different permeability of active agents presented on the different animal skins and different skin sites of the same species. Histological and biochemical properties of porcine skin have been repeatedly shown to be similar to human skin. The thickness of viable skin layers, numbers of hairs, the vascular anatomy and collagen fiber arrangement in the dermis, as well as the contents of SC glycosphingolipids and ceramides were similar in man and in
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the domestic pig (Wester & Maibach, 1989; Simon & Maibach, 2000; Jacobi et al., 2007). Due to its availability, skin of rodents was the most commonly used in in vitro and in vivo percutaneous permeation studies (Godin & Touitou, 2007). The advantages of rodents were their small size, uncomplicated handling and relatively low cost. However, a number of hairless species (nude mice, hairless mice) in which the absence of hair coat mimics the human skin better than hairy skin (Simon & Maibach, 1998). The penetration enhancing effect of Azone on various types of skin has been validated by Niazy (1996). It was interesting that the lower permeability of the skin model exhibited the more significant improvement of permeability when enhancer was applied. But the comparisons of function of skin targeting modifiers on skin models have rarely been reported. This study focused on the comparison of permeability of pig abdominal skin, pig ear skin and hairless mouse skin with and without penetration modifiers. Different influencing factors were studied and possible reasons for different results caused by the same formulation were proposed.
Materials and methods Materials and skin membranes Metronidazole (MTZ) was purchased from ALFA AESAR (ZhongAn pharmaceutical, Tianjin, China). A racemic mixture of 1,4-cyclohexanediol was purchased from SigmaAldrich (St. Louis, MO). A racemic mixture of 1,2-hexanediol was purchased from Sabina Corporation (Piscataway, NJ). A racemic mixture of 1,2-heptanediol was synthesized in our laboratories from standard dihydroxylation method of 1-heptene. KlucelÕ MF was obtained from Hercules, Inc. (Wilmington, DE). All other reagents were of analytical grade. Skin samples were taken from the pig of about 3 months old. Excised pig skin from ear and abdomen was used in our experiments. All animal protocols were performed under the guidelines for humane and responsible use of animals in research set by Tianjin University School of Pharmaceutical Science and Technology. After removing the hair and the subcutaneous fatty tissue, the skin was cleaned in normal saline, then divided into smaller pieces and stored at 20 C prior to use. Preparation of formulations Six formulations were prepared and the preparation procedure was as follows (Table 1). Take formulation F3 as the example, Table 1. Formulations containing MTZ (F1–F6) in this study.
1,4-cyclohexanediol (1%) was dissolved in a solution of 1,2hexanediol (4%) in water, MTZ (0.75%) was dispersed in the above clarify solution with a stirrer until MTZ was dissolved. The KlucelÕ MF (0.75%) was added to the solution as gelling agent. Keep on stirring until the solution was gelled. Due to the lower water solubility of 1,2-heptanediol than 1,2-hexanediol, the concentration of 1,2-heptanediol in the formulation was 1%. In vitro skin permeation studies The skin membranes were mounted on Franz Diffusion Cell (Pharmacopoeia Standard Instrument Factory, Tianjin, China) with the SC side facing upwards into the donor compartment (diffusion area ¼ 1.77 cm2) and the dermal side facing downwards into the receptor compartment (volume ¼ 17 ml). The receptor chamber was filled with PBS (pH 7.4) with a magnetic stirrer continuously stirred at 500 rpm. Before each experiment, the skin samples were thawed to room temperature and equilibrated at 32 0.1 C for 1 h with PBS (pH 7.4) in Franz Diffusion Cell. The temperature maintained and infinite doses (100 mg of the formulations, which corresponded to 750 mg of MTZ) were applied to the skin samples. The donor chamber was sealed with ParafilmÕ to inhibit evaporation of the formulations. Each set of experiments was run in six parallels. At the end of each time interval (2, 4, 8, 12, 16, 20 and 24 h), the skin surface was wiped with cotton ball soaked with PBS (pH 7.4). SC layer was removed by the tape-stripping method (Howes et al., 1996). The residual drug in the epidermis and dermis was extracted by a mixed solution of 2.5 ml methanol and 2.5 ml PBS (pH 7.4). After removal of the SC, skin was minced, vortexed with previous solution and then centrifuged, the supernatant was filtered and ready for analysis. The receptor medium was withdrawn from the receptor and ready for analysis. HPLC analytical method HPLC analysis was performed on a Thermo RP-C18 (250 mm 4.6 mm I.D., 5 mm) column. The mobile phase was a degassed and filtered (0.45 mm; Millipore) mixture of double distilled water-methanol (80:20, v/v) and eluted with a flow rate of 1.0 ml/min. Injection volume was 20 ml. UV detection was performed at 320 nm and the temperature of the column was maintained at 30 C. The analytical method was validated for linearity, precision and accuracy. The plot showed good linearity with correlation coefficient of 0.9999. LOD and LOQ were determined to be 100 and 150 ng/ml, respectively. Intraday variability was 50.2% and interday variability was also calculated to be 52.0%.
Ingredient (g/100 g) Formulation F1 F2 F3 F4 F5 F6
1,2-Hexanediol
1,2-Heptanediol
1,4-Cyclohexanediol
– – 4.0 4.0 – –
– – – – 1.0 1.0
– 1.0 1.0 – 1.0 –
Each formulation contains MTZ (0.75%) and KlucelÕ MF (0.75%). Infinite dose was 100 mg of the formulations, which correspond to 750 mg of MTZ.
Data and statistical analysis The amount of active agent in the samples was determined using validated assay method. In the in vitro permeation studies, three transdermal parameters such as mean flux, lag time (Tlag) and cumulative amount after 24 h (Q24) were used to evaluate permeability. Data analysis was carried out with Microsoft Excel (Batheja et al., 2009). Flux values were determined from slopes of plots of concentration in the receptor phase versus time curves and expressed as
Skin targeting effect of penetration modifiers
DOI: 10.3109/10717544.2013.869276
mg per cm2 per hour. Tlag standed for the duration time of drug started to penetration into receptor medium after administration. It was determined from X-axis value of concentration– time curves which mentioned above when Y-axis value was zero. The degree of topical targeting effect was defined as the targeting ratio (TR), which was calculated from the following equation:
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TR ¼ Rtext =Rcontrol
ð1Þ
where R was the skin retention, the amount of residual drug in the epidermis and dermis extracted from skin. Rtest and Rcontrol were the skin retention of the formulation containing penetration modifiers (F2–F6) and formulation without modifiers (F1) at 24 h, respectively. TR was the targeting ratio, the degree of the increased amount of drug maintaining in skin epidermis and dermis layer for therapy after addition of penetration modifiers. The values above unity represented the topical targeting effect of the formulation. Paired two-tailed Student’s t-test was performed to calculate the statistical significance (*p50.05, **p50.01). Results were expressed as mean SD (n ¼ 6 independent samples).
Results Results of six formulations in the same skin model The six tested formulations were applied to pig abdominal skin and pig ear skin, coupled with the laboratory previous data obtained by hairless mouse skin, consistent results were observed (Table 2). Take pig abdominal skin as an example, it was observed that formulation F3 has the lowest value of flux (3.63 mg/cm2/h) and Q24 (139.50 mg) compared with other ones, respectively, which standing for the notable skin targeting effect by retarding its permeability. The higher flux (7.26 mg/cm2/h) and Q24 (244.13 mg) of formulation F5 compared with F3 also revealed its topical targeting effect in a certain degree. Formulations F2, F4 and F6 (1,2-diol or Table 2. Skin permeation parameters of MTZ on various skin models. Formulation
Flux (mg/cm2/h)
Pig abdominal skin model F1 8.63 0.77 F2 9.01 0.61 F3 3.63 0.19** F4 9.27 0.51 F5 7.26 0.11* F6 9.01 0.15 Pig ear skin model F1 6.37 0.13 F2 6.56 0.21 F3 3.84 0.21* F4 6.51 0.17 F5 5.36 0.18* F6 6.81 0.08 Hairless mouse skin model F1 15.09 0.14 F2 15.15 0.29 F3 12.6 0.03* F4 14.13 0.25 F5 13.45 0.42 F6 14.20 0.27
Q24 (mg)
Tlag (h)
328.51 34.68 314.45 28.48 139.50 10.21** 333.78 22.16 244.13 34.41** 338.67 41.89
2.86 0.14 3.95 0.22 2.14 0.08 3.98 0.12 3.33 0.04 3.78 0.03
210.10 25.36 193.86 19.30 121.43 7.05** 221.33 35.55 175.61 13.68* 218.56 33.71
3.69 0.09 3.74 0.11 3.61 0.09 4.17 0.05 3.33 0.07 3.47 0.07
618.76 34.11 621.98 41.92 508.62 18.43** 574.30 12.19 539.15 11.76* 580.23 18.21
0.78 0.06 0.75 0.02 1.20 0.37 0.98 0.06 1.35 0.14 0.91 0.31
Infinite dose was 100 mg of the formulations, which correspond to 750 mg of MTZ. Each value represented the mean SD (n ¼ 6). **p50.01, *p50.05.
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1,4-cyclohexanediol appeared alone) were not significantly different from control formulation F1 (both 1,2-diol and 1,4-cyclohexanediol were absent) on various parameters. A similar percutaneous data for topically applied of MTZ was observed despite that the pig ear tissue shows similarities and also dissimilarities in skin structure to pig abdominal skin. But values of flux (3.84–6.81 mg/cm2/h) and Q24 (121– 221 mg) in pig ear skin were slightly lower than those in pig abdominal skin. In our earlier studies, the synergistic topical targeting effect of 1,4-cyclohexanediol and 1,2-hexanediol/1,2-heptanediol was proved using hairless mouse skin. Q24 was up to 622 mg, flux value was 12.6–15.1 mg/cm2/h. The results indicated the predominantly higher percutaneous absorption of MTZ in hairless mouse skin than that in pig abdominal skin and pig ear skin. Permeability of MTZ in different skin models Table 2 showed that the percutaneous penetration parameters of formulations F2, F4 and F6 had no significant differences with F1 in the same skin model (p40.5). F5 presented significant targeting effect while F3 displayed a more efficient role. Formulations did not show exactly the same results on each skin model. It was found that the steady-state flux values for formulation F1 on hairless mouse skin were much higher than those on pig abdominal skin and pig ear skin, which suggesting the higher transdermal absorption characteristics of hairless mouse skin in the same experimental conditions. It was learned that the skin permeability of MTZ in different skin models decreasing in the order: hairless mouse skin4pig abdominal skin4pig ear skin (flux values were 15.09, 8.63 and 6.37 mg/cm2/h of formulation F1, respectively). The flux values of formulation containing a combination of 1,4-cyclohexanediol and 1,2-hexanediol (F3) decreased in different degrees relative to those of control formulation (F1) through three skin types (about 20% for hairless mouse skin, 60% for pig abdominal skin and 40% for pig ear skin, respectively). The decreases of flux in three skin types were in the following order based on these data: pig abdominal skin4pig ear skin4hairless mouse skin. Although much higher values of the accumulated amounts of MTZ were observed in hairless mouse skin at each time point, the decrement of Q24 represented the same order after using synergistic penetration modifiers. Tlag in Table 2 suggested that drug penetrated fastest in hairless mouse skin and slowest in pig ear skin. The three skin models were different from each other on skin (or SC) thickness, the composition of intercellular SC lipids and the number of skin shafts. Beyond these diversities, the synergistic modifiers still presented its general skin targeting effect on species or on different application sites in the same animal model. Skin targeting effect of MTZ in different skin models As described in first part of results, only formulation F3 and F5 performed skin targeting effect due to presence of penetration modifiers while formulation F2, F4 and F6 did not show significant difference compared to control formulation (F1). F3 appeared a particularly excellent role of skin
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Hairless mouse skin
Pig abdominal skin **
Pig ear skin **
50.0% 40.0% 30.0% 20.0% 10.0% 0.0%
(B) 60.0%
2
4
8
Hairless mouse skin
12 Time/h
16
20
Pig abdominal skin **
24 Pig ear skin *
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50.0% 40.0%
Discussion
30.0% 20.0% 10.0% 0.0%
no significant difference between the two skin models (p40.05). On the contrary, skin retention in hairless mouse skin declined during the whole experiment. Data revealed significant lower drug content in hairless mouse skin than that in pig abdominal skin and pig ear skin. Skin retention in the two pig skin types both presented a same trend that MTZ accumulated in skin layers before reaching the steady-state under the effect of penetration modifiers (Figure 1B). Residual drug in hairless mouse skin continued to decrease during the administration time but not such evidently relative to that of F1. TRs of each skin models under the effect of F3 were calculated, and the results represented the topical targeting effect of synergistic penetration modifiers. The data showed the following order of topical targeting effect: hairless mouse skin4pig skin4pig ear skin, which exhibited a different order from that of flux (TRs were 1.79, 1.24 and 1.05, respectively).
2
4
8
12 Time/h
16
20
24
Figure 1. Percentage of skin retention of MTZ in various skin layers at time intervals. (A) Skin retention of MTZ in F1. (B) Skin retention of MTZ in F3. Both skin models from pig were compared with hairless mouse skin. **p50.01, *p50.05.
targeting. However F2, F4, F5 and F6 all performed differently in different skin models, when referred to skin targeting effect, we took F1 and F3 to make a comparison. The data of percentage of skin retention in pig abdominal skin and in pig ear skin were compared to those in hairless mouse skin to investigate the effects of formulation with or without penetration modifiers on transdermal delivery of MTZ (see Figure 1). Percentage of skin retention (expressed as %) was calculated by the amount of skin retention (expressed as mg) divided by total drug amount of applied formulation. The experimental data suggested that the drug delivery performed a consistent trend in all skin models under the influence of modifiers. In addition, the targeting effects gradually increased with the passage of time (i.e. the difference of skin retention exaggerated with time between F1 and F3). There was still continuing skin targeting effect even in the time point of 24 h. Overall, residual drugs on the surface increased while the accumulated amount in the collection medium significantly decreased when synergistic modifiers presented. The marked topical targeting effect performed by intercepting drug in outer skin and preventing it from permeating into systemic circulation, which might leading to toxic side effects. Figure 1(A) showed the skin retention in different skin models of F1. Skin retention of MTZ gradually gained and reached a steady-state amount at 8 h in pig abdominal skin while it remained a relative constant level throughout 24 h in pig ear skin. Paired two-tailed Student’s t-test suggested
Various animal skin models have been studied as surrogates for unavailable human skin in in vitro percutaneous studies. One of the main challenges of biopharmaceutical research was finding an appropriate animal model for prediction of percutaneous absorption in humans. The lack of correlation in transdermal permeation of molecules across species or from different application sites in the same animal model was due mainly to variations in skin (or SC) thickness, in the composition of intercellular SC lipids and in the number of skin shafts (Godin & Touitou, 2007). Pig abdominal skin, pig ear skin and hairless mouse skin tested in this study have been used for prediction of percutaneous absorption in humans in various studies. A skin targeting mechanism has been reported in the published article by our laboratory. 1,2-diol could form a complex with 1,4-cyclohexanediol, and the hydrophobic alkyl chain in 1,2-diol might intercalate into SC layer carried 1,4-cyclohexanediol to construct a two-side H-bonding with neighboring ceramide molecules. Thus MTZ could be inhibited by the cross-linked network (Li et al., 2011). The role of SC on percutaneous penetration of MTZ The role of SC thickness Differences in skin permeability among species have been compared through skin structural features such as thickness of the various layers, number of cell layers and hair follicle anatomy (Barbero & Frasch, 2009). It has been long recognized that the main barrier to percutaneous absorption was SC and its thickness increased with animal size (Niazy, 1996). The SC thickness of three skin models had been measured, the SC thickness of pig abdominal skin, pig ear skin and hairless mouse skin were approximately 25, 23 and 14 mm, respectively, which was corresponding to the data reported in the references. Studies examining thickness of various skin layers of pigs have shown that the SC thickness of pig abdominal skin was 21–26 mm which was comparable to pig ear skin (17–28 mm) (Jacobi et al., 2007). The SC thickness of pig skin was comparable with human skin (about 30 mm verified by various studies), while the SC
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thickness of hairless mouse skin was only half of human. However, the diffusional path length was much longer than the simple thickness of the SC (&20 mm) and has been estimated as long as 500 mm (Hadgraft, 2004). Data have shown that the values of flux were reduced by the high concentration of penetration modifiers. However, each of the three different skin models had a different reaction when the same concentration of penetration modifiers were applied (4% of 1,2-hexanediol and 1% of 1,4-cyclohexanediol in F3). Hairless mouse skin did not have high density of hair follicles, so that the only explanation for its higher permeability than pig abdominal skin and pig ear skin was less thickness of various layers and smaller number of cell layers. As described in permeability of MTZ in different skin models part in results, the most obvious reduction of flux values appeared in pig abdominal skin, a compact structure was constructed with the penetration modifiers in the thick SC. Drug molecules were more susceptible to be hindered by the network through such a long pathway. In contrast, the hairless mouse skin displayed inhibited effects at a very low degree from its thin SC layer, and it also took less time for molecular of MTZ to transfer through the SC layer. Thus the values of flux were much lower in hairless mouse skin compared with that in pig abdominal skin and pig ear skin. The transfer pathway through SC Previously, varied researches of attempts to relate permeability with skin structure might owe to the fact that gross or macro anatomical structural features had been studied, but recent years more attention focused on specific permeation pathways to relating micro-level structural features to skin penetration (Barbero & Frasch, 2009). Two main pathways for transdermal penetration were normally considered (although additional alternative pathways were proposed in the study of Prausnitz et al., 2004) either via SC through the intercellular lipid lamellae or through cutaneous appendages, i.e. hair follicles and sweat glands (Jacobi et al., 2006; Loan Honeywell-Nguyen et al., 2006). It was generally assumed that, under normal circumstances, the predominant route was through the intercellular spaces in the SC (Howes et al., 1996). As the mainly barrier, SC had fatty acids, esterified or unesterified sterols and ceramides filled with intercellular spaces. Janu˚sˇova´ et al. (2013) have proved the partial recovery of the skin barrier function by measure skin impedance while applied and removed enhancer. Sarah et al. (2009) also put forward that high lipophilic enhancers generally had longer duration of action than low lipophilic ones, and the enhance action would be weaken as the concentration of enhancers in SC decreased. It was reasonable to deduce that there was a same characteristic for penetration modifier molecules used in this study. The modifier molecules did not persist in the skin barrier and was relatively rapidly eliminated, probably by simple diffusion into lower epidermal layers with the passage of time. The complex of modifier molecules and lipid in SC layer would break up in this molecular movement. Penetration modifiers constantly entered SC to play the role of interception of MTZ, therefore drug gradually accumulated in SC to achieve topical skin targeting effect. Then modifier molecules
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moved into the lower dermal layer thus leading to the weakening of inhibition function because of the structure recovery of SC. Thus SC as a reservoir sustained released drug into lower layer. That was why skin retention of MTZ gradually gained at first but reached a steady-state or even declined several hours later. Selection of skin models in in vitro percutaneous studies Differences in percutaneous absorption of MTZ did exist among the skin models examined in this investigation. It was hardly to decide a unique skin as the best model used in in vitro percutaneous experiment because there was no animal skin that completely mimics the penetration characteristics of human skin. Thus it was necessary to select animal models based on specific purposes of studies. Some results showed that rodent skins were poor models for human skin (Catz & Friend, 1990), and hairless mouse skin always appeared to considerably overestimate the penetration of active agents. Systemic effect was evaluated by concentration of drugs penetrating into receptor medium in transdermal formulations. But the target substance might be too low to make a precise analysis for the efficient barrier properties of skin layers. Therefore, the excellent permeability of hairless mouse skin made it as a suitable model for transdermal formulations. In aspect to topical formulations, maximal skin retention and minimal skin penetration was expected. Thus the pig abdominal skin and pig ear skin with a thick SC layer were probably the better choice. However, selection criteria for skins turned out to be different in formulations with penetration modifiers. Pig abdominal skin revealed the most significant decrease of permeability under the role of penetration modifiers when estimated by flux value and Q24. On the contrast, TR suggested that hairless mouse skin had the highest increase of skin retention after administration of formulations containing penetration modifiers. An available direction was determined depend on the results mentioned above. Pig abdominal skin was an effective model in forecasting the impact on permeability in transdermal delivery system. As presence of penetration modifiers, hairless mouse skin became a rational prediction about the effects of topical modifiers, particularly when estimating the skin retention.
Conclusions This study further verified the skin targeting effects of formulations of MTZ containing a combination penetration modifiers of 1,2-hexanediol (or 1,2-heptanediol) and 1,4cyclohexanediol. The penetration modifiers worked in various degrees in different skin models for their diverse anatomical features. Speaking concretely, the most reduction of flux appeared in the pig abdominal skin whereas the most increment of skin retention observed in hairless mouse skin compared to control formulation. However, MTZ was stored up in SC and sustained released to deeper layers, it appeared a significant lower content in receptor medium under the influence of penetration modifiers. Selection criteria of skin models have been discussed in this study, hairless mouse skin and pig abdominal skin were considered to be available
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models for estimating the alteration of skin retention and flux value under the influence of penetration modifiers, respectively.
Declaration of interest The authors declare that there are no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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References Asbill CS, Michniak BB. (2000). Percutaneous penetration enhancers: local versus transdermal activity. Pharma Sci Technol Today 3: 36–41. Barbero AM, Frasch HF. (2009). Pig and guinea pig skin as surrogates for human in vitro penetration studies: a quantitative review. Toxicol In Vitro 23:1–13. Batheja P, Song Y, Wertz P, Michniak-Kohn B. (2009). Effects of growth conditions on the barrier properties of ahumanskin equivalent. Pharma Res 26:1689–700. Catz P, Friend DR. (1990). Transdermal delivery of levonorgestrel. VIII. Effect of enhancers on rat skin, hairless mouse skin, hairless guinea pig skin, and human skin. Int J Pharma 58:93–102. Doh HJ, Cho WJ, Yong CS, et al. (2003). Synthesis and evaluation of ketorolac ester prodrugs for transdermal delivery. J Pharma Sci 92: 1008–17. Godin B, Touitou E. (2007). Transdermal skin delivery: predictions for humans from in vivo, ex vivo and animal models. Adv Drug Deliv Rev 59:1152–61. Howes D, Guy R, Hadgraft J. (1996). Methods for assessing percutaneous absorption. The report and recommendations of ECVAM Workshop. Altern Lab Anim 24:81–106. Hadgraft J, Peck J, Williams DG, et al. (1996). Mechanisms of action of skin penetration enhancers/retarders: azone and analogues. Int J Pharma 141:17–25. Hadgraft J. (2004). Skin deep. Eur J Pharma Biopharma 58:291–9. Hsieh PW, Al-Suwayeh SA, Fang CL, et al. (2012). The co-drug of conjugated hydroquinone and azelaic acid to enhance topical skin targeting and decrease penetration through the skin. Eur J Pharma Biopharma 81:369–78. Janu˚sˇova´ B, Skolova´ B, Tu¨ko¨rova´ K, et al. (2013). Amino acid derivatives as transdermal permeation enhancers. J Control Release 165:91–100.
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Jacobi U, Tassopoulos T, Surber C, Lademann J. (2006). Cutaneous distribution and localization of dyes affected by vehicles all with different lipophilicity. Arch Dermatol Res 297:303–10. Jacobi U, Kaiser M, Toll R, et al. (2007). Porcine ear skin: an in vitro model for human skin. Skin Res Technol 13:19–24. Loan Honeywell-Nguyen P, Wouter Groenink HW, Bouwstra JA. (2006). Elastic vesicles as a tool for dermal and transdermal delivery. J Liposome Res 16:273–80. Li N, Su Q, Tan FP, Zhang J. (2010). Effect of 1,4-cyclohexanediol on percutaneous absorption and penetration of azelaic acid. Int J Pharma 387:167–71. Li N, Jia WB, Zhang Y, et al. (2011). Synergistic effect of 1,4cyclohexanediol and 1,2-hexanediol on percutaneous absorption and penetration of metronidazole. Int J Pharma 415:169–74. Niazy EM. (1996). Differences in excised rabbit, penetration-enhancing effect of Azone through rat, hairless mouse, guinea pig and human skins. Int J Pharma 130:225–30. Prausnitz MR, Mitragotri S, Langer R. (2004). Current status and future potential of transdermal drug delivery. Nat Rev Drug Discov 3: 115–24. Qiu YQ, Gao YH, Hu KJ, Li F. (2008). Enhancement of skin permeation of docetaxel: a novel approach combining microneedle and elastic liposomes. J Control Release 129:144–50. Rossetti FC, Lopes LB, Carollo ARH, et al. (2011). A delivery system to avoid self-aggregation and to improve in vitro and in vivo skin delivery of a phthalocyanine derivative used in the photodynamic therapy. J Control Release 155:400–8. Simon GA, Maibach HI. (1998). Relevance of hairless mouse as an experimental model of percutaneous penetration in man. Skin Pharmacol Appl Skin Physiol 11:80–6. Simon GA, Maibach HI. (2000). The pig as an experimental animal model of percutaneous permeation in man: qualitative and quantitative observations-an overview. Skin Pharmacol Appl Skin Physiol 13: 229–34. Sarah A, Ibrahim S, Li K. (2009). Effects of solvent deposited enhancers on transdermal permeation and their relationship with Emax. J Control Release 136:117–24. S ¸ enyig˘it T, Sonvico F, Barbieri S, et al. (2010). Lecithin/chitosan nanoparticles of clobetasol-17-propionate capable of accumulation in pig skin. J Control Release 142:368–73. Tsai YH, Lee KF, Huang YB, et al. (2010). In vitro permeation and in vivo whitening effect of topical hesperetin microemulsion delivery system. Int J Pharma 388:257–62. Wester RC, Maibach HI. (1989). In vivo methods for percutaneous absorption measurements. In: Brounaugh RL, Maibach HI, eds. Percutaneous absorption: mechanisms-methodology-drug delivery, 2nd edn. New York: Marcel Dekker Inc., 215–37.
ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20
Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin Meng Yu, Fang Guo, Ying Ling, Nan Li & Fengping Tan To cite this article: Meng Yu, Fang Guo, Ying Ling, Nan Li & Fengping Tan (2013): Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin, Drug Delivery To link to this article: http://dx.doi.org/10.3109/10717544.2013.869276
Published online: 13 Dec 2013.
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Date: 05 November 2015, At: 21:56
http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Delivery, Early Online: 1–6 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.869276
Topical skin targeting effect of penetration modifiers on hairless mouse skin, pig abdominal skin and pig ear skin Meng Yu, Fang Guo, Ying Ling, Nan Li, and Fengping Tan
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Tianjin Key Laboratory of Drug Delivery and High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, PR China
Abstract
Keywords
Objective: This study was to investigate the topical skin targeting effects and mechanism of combination penetration modifiers of 1,2-hexanediol (or 1,2-heptanediol) and 1,4-cyclohexanediol on transdermal absorption of metronidazole (MTZ) in different skin models. Methods: Six formulations were applied to pig abdominal skin and pig ear skin models, respectively, and the results were compared with the previous data on hairless mouse skin worked out by our laboratory. Four parameters (flux, Tlag, Q24 and targeting ratio) were used to evaluate permeability and targeting effect in skin. Results: The combined penetration modifiers played a general role on decreasing permeability without reducing skin retention. The most significant skin permeability decrement to MTZ was pig abdominal skin (permeability decrement was 20% for hairless mouse skin, 60% for pig abdominal skin and 40% for pig ear skin, respectively) while the strongest skin targeting effect appeared in hairless mouse skin (targeting ratios were 1.79 for hairless mouse skin, 1.24 for pig abdominal skin and 1.05 for pig ear skin, respectively) under the role of penetration modifiers. Conclusions: Thickness of stratum corneum (SC) was the major factor impact on skin targeting effect. Selection criteria of skin models also have been discussed in this study.
Animal skin model, penetration modifier, skin targeting, transdermal absorption, transfer pathway
Introduction Transdermal drug delivery is a non-invasive and user-friendly delivery method. Traditionally, transdermal formulation was designed to achieve systemic therapeutics by transfering drug into blood circulation through skin layers. Physical and chemical methods have been developed to overcome the remarkable barrier properties of the outermost layer of skin, the stratum corneum (SC) and enhance the transdermal delivery of drugs (Doh et al., 2003; Qiu et al., 2008; Rossetti et al., 2011). However, transdermal formulation attracted much attention on topical skin targeting application in recent decades. Active agents from applied formulations were able to penetrate the SC layer and reach dermatologically viable layer to play a therapeutic role. However they were also able to penetrate into systematic circulation and lead to toxic side effects. In this study, a nitroimidazole derivative metronidazole (MTZ) was used as a model drug, which was expected to skin targeting for therapy of acne and rosacea. MTZ was an acidic drug with pKa 2.6, it was able to penetrate the skin effectively for its small molecular weight (Mw ¼ 171.15 g/mol), but it did not indicating potential retention within lipid domains and the establishment of a reservoir for its low lipophicity (logP ¼ 0.18). For treatment of dermatological conditions, an ideal topical formulation is to impart maximal skin Address for correspondence: Nan Li, Tianjin Key Laboratory of Drug Delivery and High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, 300072 Tianjin, PR China. Tel: þ86 022 27404986. Email: [email protected]
History Received 16 August 2013 Accepted 21 November 2013
retention and minimal systematic penetration. Thus, there is a significant need in discovering safe and effective skin targeting measures. Varying measures have been published to enhance skin retention, such as making prodrugs (Hsieh et al., 2012), using vehicles (¸Senyig˘it et al., 2010; Tsai et al., 2010) and adding penetration modifiers. Adding penetration modifiers has significant advantages over other measures for ease of manipulation, available without specific devices. A plenty of researches have been done to improve penetration modifiers. Hadgraft et al. (1996) have reported that some Azone derivative might inhibit penetration of MTZ efficiently. Asbill & Michniak (2000) successfully prevented drug from transfering into deeper skin layers by using chemical skin modifiers. In the previous study of our laboratory, skin targeting effects of 1,4-cyclohexanediol alone and synergistic with 1,2-hexanediol have been validated (Li et al., 2010, 2011). Although the effect of penetration modifiers have been investigated in plenty of researches, various animal skin models were used depended on different purposes. There was not a specifically animal skin model as a surrogate for human in in vitro penetration studies. Different permeability of active agents presented on the different animal skins and different skin sites of the same species. Histological and biochemical properties of porcine skin have been repeatedly shown to be similar to human skin. The thickness of viable skin layers, numbers of hairs, the vascular anatomy and collagen fiber arrangement in the dermis, as well as the contents of SC glycosphingolipids and ceramides were similar in man and in
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the domestic pig (Wester & Maibach, 1989; Simon & Maibach, 2000; Jacobi et al., 2007). Due to its availability, skin of rodents was the most commonly used in in vitro and in vivo percutaneous permeation studies (Godin & Touitou, 2007). The advantages of rodents were their small size, uncomplicated handling and relatively low cost. However, a number of hairless species (nude mice, hairless mice) in which the absence of hair coat mimics the human skin better than hairy skin (Simon & Maibach, 1998). The penetration enhancing effect of Azone on various types of skin has been validated by Niazy (1996). It was interesting that the lower permeability of the skin model exhibited the more significant improvement of permeability when enhancer was applied. But the comparisons of function of skin targeting modifiers on skin models have rarely been reported. This study focused on the comparison of permeability of pig abdominal skin, pig ear skin and hairless mouse skin with and without penetration modifiers. Different influencing factors were studied and possible reasons for different results caused by the same formulation were proposed.
Materials and methods Materials and skin membranes Metronidazole (MTZ) was purchased from ALFA AESAR (ZhongAn pharmaceutical, Tianjin, China). A racemic mixture of 1,4-cyclohexanediol was purchased from SigmaAldrich (St. Louis, MO). A racemic mixture of 1,2-hexanediol was purchased from Sabina Corporation (Piscataway, NJ). A racemic mixture of 1,2-heptanediol was synthesized in our laboratories from standard dihydroxylation method of 1-heptene. KlucelÕ MF was obtained from Hercules, Inc. (Wilmington, DE). All other reagents were of analytical grade. Skin samples were taken from the pig of about 3 months old. Excised pig skin from ear and abdomen was used in our experiments. All animal protocols were performed under the guidelines for humane and responsible use of animals in research set by Tianjin University School of Pharmaceutical Science and Technology. After removing the hair and the subcutaneous fatty tissue, the skin was cleaned in normal saline, then divided into smaller pieces and stored at 20 C prior to use. Preparation of formulations Six formulations were prepared and the preparation procedure was as follows (Table 1). Take formulation F3 as the example, Table 1. Formulations containing MTZ (F1–F6) in this study.
1,4-cyclohexanediol (1%) was dissolved in a solution of 1,2hexanediol (4%) in water, MTZ (0.75%) was dispersed in the above clarify solution with a stirrer until MTZ was dissolved. The KlucelÕ MF (0.75%) was added to the solution as gelling agent. Keep on stirring until the solution was gelled. Due to the lower water solubility of 1,2-heptanediol than 1,2-hexanediol, the concentration of 1,2-heptanediol in the formulation was 1%. In vitro skin permeation studies The skin membranes were mounted on Franz Diffusion Cell (Pharmacopoeia Standard Instrument Factory, Tianjin, China) with the SC side facing upwards into the donor compartment (diffusion area ¼ 1.77 cm2) and the dermal side facing downwards into the receptor compartment (volume ¼ 17 ml). The receptor chamber was filled with PBS (pH 7.4) with a magnetic stirrer continuously stirred at 500 rpm. Before each experiment, the skin samples were thawed to room temperature and equilibrated at 32 0.1 C for 1 h with PBS (pH 7.4) in Franz Diffusion Cell. The temperature maintained and infinite doses (100 mg of the formulations, which corresponded to 750 mg of MTZ) were applied to the skin samples. The donor chamber was sealed with ParafilmÕ to inhibit evaporation of the formulations. Each set of experiments was run in six parallels. At the end of each time interval (2, 4, 8, 12, 16, 20 and 24 h), the skin surface was wiped with cotton ball soaked with PBS (pH 7.4). SC layer was removed by the tape-stripping method (Howes et al., 1996). The residual drug in the epidermis and dermis was extracted by a mixed solution of 2.5 ml methanol and 2.5 ml PBS (pH 7.4). After removal of the SC, skin was minced, vortexed with previous solution and then centrifuged, the supernatant was filtered and ready for analysis. The receptor medium was withdrawn from the receptor and ready for analysis. HPLC analytical method HPLC analysis was performed on a Thermo RP-C18 (250 mm 4.6 mm I.D., 5 mm) column. The mobile phase was a degassed and filtered (0.45 mm; Millipore) mixture of double distilled water-methanol (80:20, v/v) and eluted with a flow rate of 1.0 ml/min. Injection volume was 20 ml. UV detection was performed at 320 nm and the temperature of the column was maintained at 30 C. The analytical method was validated for linearity, precision and accuracy. The plot showed good linearity with correlation coefficient of 0.9999. LOD and LOQ were determined to be 100 and 150 ng/ml, respectively. Intraday variability was 50.2% and interday variability was also calculated to be 52.0%.
Ingredient (g/100 g) Formulation F1 F2 F3 F4 F5 F6
1,2-Hexanediol
1,2-Heptanediol
1,4-Cyclohexanediol
– – 4.0 4.0 – –
– – – – 1.0 1.0
– 1.0 1.0 – 1.0 –
Each formulation contains MTZ (0.75%) and KlucelÕ MF (0.75%). Infinite dose was 100 mg of the formulations, which correspond to 750 mg of MTZ.
Data and statistical analysis The amount of active agent in the samples was determined using validated assay method. In the in vitro permeation studies, three transdermal parameters such as mean flux, lag time (Tlag) and cumulative amount after 24 h (Q24) were used to evaluate permeability. Data analysis was carried out with Microsoft Excel (Batheja et al., 2009). Flux values were determined from slopes of plots of concentration in the receptor phase versus time curves and expressed as
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DOI: 10.3109/10717544.2013.869276
mg per cm2 per hour. Tlag standed for the duration time of drug started to penetration into receptor medium after administration. It was determined from X-axis value of concentration– time curves which mentioned above when Y-axis value was zero. The degree of topical targeting effect was defined as the targeting ratio (TR), which was calculated from the following equation:
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TR ¼ Rtext =Rcontrol
ð1Þ
where R was the skin retention, the amount of residual drug in the epidermis and dermis extracted from skin. Rtest and Rcontrol were the skin retention of the formulation containing penetration modifiers (F2–F6) and formulation without modifiers (F1) at 24 h, respectively. TR was the targeting ratio, the degree of the increased amount of drug maintaining in skin epidermis and dermis layer for therapy after addition of penetration modifiers. The values above unity represented the topical targeting effect of the formulation. Paired two-tailed Student’s t-test was performed to calculate the statistical significance (*p50.05, **p50.01). Results were expressed as mean SD (n ¼ 6 independent samples).
Results Results of six formulations in the same skin model The six tested formulations were applied to pig abdominal skin and pig ear skin, coupled with the laboratory previous data obtained by hairless mouse skin, consistent results were observed (Table 2). Take pig abdominal skin as an example, it was observed that formulation F3 has the lowest value of flux (3.63 mg/cm2/h) and Q24 (139.50 mg) compared with other ones, respectively, which standing for the notable skin targeting effect by retarding its permeability. The higher flux (7.26 mg/cm2/h) and Q24 (244.13 mg) of formulation F5 compared with F3 also revealed its topical targeting effect in a certain degree. Formulations F2, F4 and F6 (1,2-diol or Table 2. Skin permeation parameters of MTZ on various skin models. Formulation
Flux (mg/cm2/h)
Pig abdominal skin model F1 8.63 0.77 F2 9.01 0.61 F3 3.63 0.19** F4 9.27 0.51 F5 7.26 0.11* F6 9.01 0.15 Pig ear skin model F1 6.37 0.13 F2 6.56 0.21 F3 3.84 0.21* F4 6.51 0.17 F5 5.36 0.18* F6 6.81 0.08 Hairless mouse skin model F1 15.09 0.14 F2 15.15 0.29 F3 12.6 0.03* F4 14.13 0.25 F5 13.45 0.42 F6 14.20 0.27
Q24 (mg)
Tlag (h)
328.51 34.68 314.45 28.48 139.50 10.21** 333.78 22.16 244.13 34.41** 338.67 41.89
2.86 0.14 3.95 0.22 2.14 0.08 3.98 0.12 3.33 0.04 3.78 0.03
210.10 25.36 193.86 19.30 121.43 7.05** 221.33 35.55 175.61 13.68* 218.56 33.71
3.69 0.09 3.74 0.11 3.61 0.09 4.17 0.05 3.33 0.07 3.47 0.07
618.76 34.11 621.98 41.92 508.62 18.43** 574.30 12.19 539.15 11.76* 580.23 18.21
0.78 0.06 0.75 0.02 1.20 0.37 0.98 0.06 1.35 0.14 0.91 0.31
Infinite dose was 100 mg of the formulations, which correspond to 750 mg of MTZ. Each value represented the mean SD (n ¼ 6). **p50.01, *p50.05.
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1,4-cyclohexanediol appeared alone) were not significantly different from control formulation F1 (both 1,2-diol and 1,4-cyclohexanediol were absent) on various parameters. A similar percutaneous data for topically applied of MTZ was observed despite that the pig ear tissue shows similarities and also dissimilarities in skin structure to pig abdominal skin. But values of flux (3.84–6.81 mg/cm2/h) and Q24 (121– 221 mg) in pig ear skin were slightly lower than those in pig abdominal skin. In our earlier studies, the synergistic topical targeting effect of 1,4-cyclohexanediol and 1,2-hexanediol/1,2-heptanediol was proved using hairless mouse skin. Q24 was up to 622 mg, flux value was 12.6–15.1 mg/cm2/h. The results indicated the predominantly higher percutaneous absorption of MTZ in hairless mouse skin than that in pig abdominal skin and pig ear skin. Permeability of MTZ in different skin models Table 2 showed that the percutaneous penetration parameters of formulations F2, F4 and F6 had no significant differences with F1 in the same skin model (p40.5). F5 presented significant targeting effect while F3 displayed a more efficient role. Formulations did not show exactly the same results on each skin model. It was found that the steady-state flux values for formulation F1 on hairless mouse skin were much higher than those on pig abdominal skin and pig ear skin, which suggesting the higher transdermal absorption characteristics of hairless mouse skin in the same experimental conditions. It was learned that the skin permeability of MTZ in different skin models decreasing in the order: hairless mouse skin4pig abdominal skin4pig ear skin (flux values were 15.09, 8.63 and 6.37 mg/cm2/h of formulation F1, respectively). The flux values of formulation containing a combination of 1,4-cyclohexanediol and 1,2-hexanediol (F3) decreased in different degrees relative to those of control formulation (F1) through three skin types (about 20% for hairless mouse skin, 60% for pig abdominal skin and 40% for pig ear skin, respectively). The decreases of flux in three skin types were in the following order based on these data: pig abdominal skin4pig ear skin4hairless mouse skin. Although much higher values of the accumulated amounts of MTZ were observed in hairless mouse skin at each time point, the decrement of Q24 represented the same order after using synergistic penetration modifiers. Tlag in Table 2 suggested that drug penetrated fastest in hairless mouse skin and slowest in pig ear skin. The three skin models were different from each other on skin (or SC) thickness, the composition of intercellular SC lipids and the number of skin shafts. Beyond these diversities, the synergistic modifiers still presented its general skin targeting effect on species or on different application sites in the same animal model. Skin targeting effect of MTZ in different skin models As described in first part of results, only formulation F3 and F5 performed skin targeting effect due to presence of penetration modifiers while formulation F2, F4 and F6 did not show significant difference compared to control formulation (F1). F3 appeared a particularly excellent role of skin
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Hairless mouse skin
Pig abdominal skin **
Pig ear skin **
50.0% 40.0% 30.0% 20.0% 10.0% 0.0%
(B) 60.0%
2
4
8
Hairless mouse skin
12 Time/h
16
20
Pig abdominal skin **
24 Pig ear skin *
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50.0% 40.0%
Discussion
30.0% 20.0% 10.0% 0.0%
no significant difference between the two skin models (p40.05). On the contrary, skin retention in hairless mouse skin declined during the whole experiment. Data revealed significant lower drug content in hairless mouse skin than that in pig abdominal skin and pig ear skin. Skin retention in the two pig skin types both presented a same trend that MTZ accumulated in skin layers before reaching the steady-state under the effect of penetration modifiers (Figure 1B). Residual drug in hairless mouse skin continued to decrease during the administration time but not such evidently relative to that of F1. TRs of each skin models under the effect of F3 were calculated, and the results represented the topical targeting effect of synergistic penetration modifiers. The data showed the following order of topical targeting effect: hairless mouse skin4pig skin4pig ear skin, which exhibited a different order from that of flux (TRs were 1.79, 1.24 and 1.05, respectively).
2
4
8
12 Time/h
16
20
24
Figure 1. Percentage of skin retention of MTZ in various skin layers at time intervals. (A) Skin retention of MTZ in F1. (B) Skin retention of MTZ in F3. Both skin models from pig were compared with hairless mouse skin. **p50.01, *p50.05.
targeting. However F2, F4, F5 and F6 all performed differently in different skin models, when referred to skin targeting effect, we took F1 and F3 to make a comparison. The data of percentage of skin retention in pig abdominal skin and in pig ear skin were compared to those in hairless mouse skin to investigate the effects of formulation with or without penetration modifiers on transdermal delivery of MTZ (see Figure 1). Percentage of skin retention (expressed as %) was calculated by the amount of skin retention (expressed as mg) divided by total drug amount of applied formulation. The experimental data suggested that the drug delivery performed a consistent trend in all skin models under the influence of modifiers. In addition, the targeting effects gradually increased with the passage of time (i.e. the difference of skin retention exaggerated with time between F1 and F3). There was still continuing skin targeting effect even in the time point of 24 h. Overall, residual drugs on the surface increased while the accumulated amount in the collection medium significantly decreased when synergistic modifiers presented. The marked topical targeting effect performed by intercepting drug in outer skin and preventing it from permeating into systemic circulation, which might leading to toxic side effects. Figure 1(A) showed the skin retention in different skin models of F1. Skin retention of MTZ gradually gained and reached a steady-state amount at 8 h in pig abdominal skin while it remained a relative constant level throughout 24 h in pig ear skin. Paired two-tailed Student’s t-test suggested
Various animal skin models have been studied as surrogates for unavailable human skin in in vitro percutaneous studies. One of the main challenges of biopharmaceutical research was finding an appropriate animal model for prediction of percutaneous absorption in humans. The lack of correlation in transdermal permeation of molecules across species or from different application sites in the same animal model was due mainly to variations in skin (or SC) thickness, in the composition of intercellular SC lipids and in the number of skin shafts (Godin & Touitou, 2007). Pig abdominal skin, pig ear skin and hairless mouse skin tested in this study have been used for prediction of percutaneous absorption in humans in various studies. A skin targeting mechanism has been reported in the published article by our laboratory. 1,2-diol could form a complex with 1,4-cyclohexanediol, and the hydrophobic alkyl chain in 1,2-diol might intercalate into SC layer carried 1,4-cyclohexanediol to construct a two-side H-bonding with neighboring ceramide molecules. Thus MTZ could be inhibited by the cross-linked network (Li et al., 2011). The role of SC on percutaneous penetration of MTZ The role of SC thickness Differences in skin permeability among species have been compared through skin structural features such as thickness of the various layers, number of cell layers and hair follicle anatomy (Barbero & Frasch, 2009). It has been long recognized that the main barrier to percutaneous absorption was SC and its thickness increased with animal size (Niazy, 1996). The SC thickness of three skin models had been measured, the SC thickness of pig abdominal skin, pig ear skin and hairless mouse skin were approximately 25, 23 and 14 mm, respectively, which was corresponding to the data reported in the references. Studies examining thickness of various skin layers of pigs have shown that the SC thickness of pig abdominal skin was 21–26 mm which was comparable to pig ear skin (17–28 mm) (Jacobi et al., 2007). The SC thickness of pig skin was comparable with human skin (about 30 mm verified by various studies), while the SC
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thickness of hairless mouse skin was only half of human. However, the diffusional path length was much longer than the simple thickness of the SC (&20 mm) and has been estimated as long as 500 mm (Hadgraft, 2004). Data have shown that the values of flux were reduced by the high concentration of penetration modifiers. However, each of the three different skin models had a different reaction when the same concentration of penetration modifiers were applied (4% of 1,2-hexanediol and 1% of 1,4-cyclohexanediol in F3). Hairless mouse skin did not have high density of hair follicles, so that the only explanation for its higher permeability than pig abdominal skin and pig ear skin was less thickness of various layers and smaller number of cell layers. As described in permeability of MTZ in different skin models part in results, the most obvious reduction of flux values appeared in pig abdominal skin, a compact structure was constructed with the penetration modifiers in the thick SC. Drug molecules were more susceptible to be hindered by the network through such a long pathway. In contrast, the hairless mouse skin displayed inhibited effects at a very low degree from its thin SC layer, and it also took less time for molecular of MTZ to transfer through the SC layer. Thus the values of flux were much lower in hairless mouse skin compared with that in pig abdominal skin and pig ear skin. The transfer pathway through SC Previously, varied researches of attempts to relate permeability with skin structure might owe to the fact that gross or macro anatomical structural features had been studied, but recent years more attention focused on specific permeation pathways to relating micro-level structural features to skin penetration (Barbero & Frasch, 2009). Two main pathways for transdermal penetration were normally considered (although additional alternative pathways were proposed in the study of Prausnitz et al., 2004) either via SC through the intercellular lipid lamellae or through cutaneous appendages, i.e. hair follicles and sweat glands (Jacobi et al., 2006; Loan Honeywell-Nguyen et al., 2006). It was generally assumed that, under normal circumstances, the predominant route was through the intercellular spaces in the SC (Howes et al., 1996). As the mainly barrier, SC had fatty acids, esterified or unesterified sterols and ceramides filled with intercellular spaces. Janu˚sˇova´ et al. (2013) have proved the partial recovery of the skin barrier function by measure skin impedance while applied and removed enhancer. Sarah et al. (2009) also put forward that high lipophilic enhancers generally had longer duration of action than low lipophilic ones, and the enhance action would be weaken as the concentration of enhancers in SC decreased. It was reasonable to deduce that there was a same characteristic for penetration modifier molecules used in this study. The modifier molecules did not persist in the skin barrier and was relatively rapidly eliminated, probably by simple diffusion into lower epidermal layers with the passage of time. The complex of modifier molecules and lipid in SC layer would break up in this molecular movement. Penetration modifiers constantly entered SC to play the role of interception of MTZ, therefore drug gradually accumulated in SC to achieve topical skin targeting effect. Then modifier molecules
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moved into the lower dermal layer thus leading to the weakening of inhibition function because of the structure recovery of SC. Thus SC as a reservoir sustained released drug into lower layer. That was why skin retention of MTZ gradually gained at first but reached a steady-state or even declined several hours later. Selection of skin models in in vitro percutaneous studies Differences in percutaneous absorption of MTZ did exist among the skin models examined in this investigation. It was hardly to decide a unique skin as the best model used in in vitro percutaneous experiment because there was no animal skin that completely mimics the penetration characteristics of human skin. Thus it was necessary to select animal models based on specific purposes of studies. Some results showed that rodent skins were poor models for human skin (Catz & Friend, 1990), and hairless mouse skin always appeared to considerably overestimate the penetration of active agents. Systemic effect was evaluated by concentration of drugs penetrating into receptor medium in transdermal formulations. But the target substance might be too low to make a precise analysis for the efficient barrier properties of skin layers. Therefore, the excellent permeability of hairless mouse skin made it as a suitable model for transdermal formulations. In aspect to topical formulations, maximal skin retention and minimal skin penetration was expected. Thus the pig abdominal skin and pig ear skin with a thick SC layer were probably the better choice. However, selection criteria for skins turned out to be different in formulations with penetration modifiers. Pig abdominal skin revealed the most significant decrease of permeability under the role of penetration modifiers when estimated by flux value and Q24. On the contrast, TR suggested that hairless mouse skin had the highest increase of skin retention after administration of formulations containing penetration modifiers. An available direction was determined depend on the results mentioned above. Pig abdominal skin was an effective model in forecasting the impact on permeability in transdermal delivery system. As presence of penetration modifiers, hairless mouse skin became a rational prediction about the effects of topical modifiers, particularly when estimating the skin retention.
Conclusions This study further verified the skin targeting effects of formulations of MTZ containing a combination penetration modifiers of 1,2-hexanediol (or 1,2-heptanediol) and 1,4cyclohexanediol. The penetration modifiers worked in various degrees in different skin models for their diverse anatomical features. Speaking concretely, the most reduction of flux appeared in the pig abdominal skin whereas the most increment of skin retention observed in hairless mouse skin compared to control formulation. However, MTZ was stored up in SC and sustained released to deeper layers, it appeared a significant lower content in receptor medium under the influence of penetration modifiers. Selection criteria of skin models have been discussed in this study, hairless mouse skin and pig abdominal skin were considered to be available
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models for estimating the alteration of skin retention and flux value under the influence of penetration modifiers, respectively.
Declaration of interest The authors declare that there are no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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