Mechanistic Studies of Permeation Enhancers

Fig. 7.1

Chemical structures of the enhancers. R, R′ = alkyl chain

Corticosterone (CS) was the main model permeant. Estradiol (ES) and hydrocortisone (HC) were the two other steroidal permeants tested in the studies. Other permeants used will be discussed in the section on “Effects of Permeation Enhancement on Permeants of Different Molecular Sizes.” The steroidal permeant at radiotracer levels and at concentrations far below its solubility was added to the donor chamber following enhancer equilibration. Samples were withdrawn from the donor and receiver chambers at predetermined time intervals and analyzed. Permeability experiments with model ionic polar permeant tetraethylammonium ion (TEA) were conducted in essentially the same manner. The total permeability coefficients (P _T) were determined from data obtained under steady-state conditions (experiment time points around three to five times longer than the lag times). The permeability coefficient of the dermis–epidermis combination (P _D/E) was obtained in the same manner, but the skin was stripped 30 times with 3 M Scotch tape prior to excision and assembly into the diffusion cell.

Another method to evaluate the potency of permeation enhancers, particularly highly lipophilic enhancers, is the direct equilibration of the skin with liquid enhancers and the subsequent skin transport experiments with PBS as the donor and receiver solution in a side-by-side diffusion cell in the absence of cosolvents or solubilizing agents (Ibrahim and Li 2009a, b). With highly lipophilic enhancers, the depletion of the enhancers from the skin to the aqueous solution in the diffusion cell chambers is expected to be minimal, and essentially constant enhancer concentration can be maintained in the SC over the duration of the permeability experiments. This method was recently used to study the structure–enhancement relationship of highly lipophilic chemical enhancers and evaluate enhancer effectiveness under the condition when the SC was saturated with the enhancer at thermodynamic activity equivalent to its pure state (i.e., at the solubility of the enhancer in the SC lipids). A disadvantage of this approach is the inability to control the enhancer concentration in the diffusion cell chamber (i.e., always at saturation in the aqueous solution) and hence in the skin. In other words, this approach cannot be used to evaluate the effects of enhancer concentration upon skin permeation enhancement. Permeation enhancer studies using this approach (Ibrahim and Li 2009a, b; Chantasart and Li 2012) will not be discussed in this chapter but have been discussed in a recent book chapter (Li and Higuchi 2015).

7.2.2.2 Reversibility Study

Diffusion cells were assembled with full thickness HMS as described for a typical permeation experiment under the symmetric and equilibrium configuration, and equilibrium between the membrane and the enhancer solution was allowed to take place. However, in this protocol, both chambers of the diffusion cell were then rinsed with PBS to remove the enhancer equilibrated in the membrane. Following the PBS rinsing regime, transport studies were carried out with PBS in both chambers. The permeability coefficients obtained with PBS after pretreatment with enhancers were then compared with those obtained with pretreatment with PBS only. Except for the highly lipophilic enhancers, all enhancers were tested for reversibility at enhancer concentrations up to enhancement factor (E) at E = 10 (Warner et al. 2001, 2003; He et al. 2003, 2004; Chantasart et al. 2004), and their effects upon permeation across SC were shown to be essentially reversible (permeability coefficients in PBS after enhancer pretreatment were within a factor of 2 of those in PBS without enhancer pretreatment).

7.2.2.3 Model Description and Analysis of Experimental Data

The permeability coefficient (P) of a probe permeant was calculated according to Eq. (7.1) (Warner et al. 2001):

$P=\frac{1}{A{ C}_{\mathrm{D}}}\frac{dQ}{dt}$

(7.1)

where A is the diffusional area of the diffusion cell, C _D is the concentration in the donor chamber, and dQ/dt is the slope of the linear region of the cumulative amount of permeant in receiver chamber (Q) vs. time plot.

Total permeability coefficient expression for full-thickness skin is written as follows:

${P}_{\mathrm{T}}=\frac{1}{\frac{1}{P_{\mathrm{SC}}}+\frac{1}{P_{\mathrm{D}/\mathrm{E}}}}$

(7.2)

where P _SC is the permeability coefficient for the stratum corneum (SC) and P _D/E is the permeability coefficient for the epidermis-dermis combination (D/E) and can be obtained from experiments of tape-stripped skin. P _SC can be further divided into parallel lipoidal and pore pathway components in SC via the following equation:

${P}_{\mathrm{SC}}={P}_{\mathrm{L}}+{P}_{\mathrm{P}}$

(7.3)

where P _L and P _P are the permeability coefficients for the lipoidal pathway and the pore pathway (TEA is used as the probe permeant for estimating the magnitude of P _P), respectively, in the SC. The intercellular lipid domain in SC is generally accepted as the lipoidal transport pathway across SC. Substituting Eq. (7.3) into Eq. (7.2) yields:

${P}_{\mathrm{T}}=\frac{1}{\frac{1}{P_{\mathrm{L}}+{P}_{\mathrm{P}}}+\frac{1}{P_{\mathrm{D}/\mathrm{E}}}}$

(7.4)

Based on the results from previous studies, the use of CS as the probe permeant allows Eq. (7.4) to be approximated by:

${P}_{\mathrm{T}}\approx {P}_{\mathrm{L}}$

(7.5)

For other steroidal permeants, P _L can be calculated by Eq. (7.4) with P _D/E and P _P values obtained from transport experiments with stripped skin and TEA, respectively. The equation for the lipoidal pathway transport enhancement factor (E) is:

$E=\frac{P_{\mathrm{L},\mathrm{X}}}{P_{\mathrm{L},\mathrm{O}}}\frac{S_{\mathrm{X}}}{S_{\mathrm{O}}}$

(7.6)

where P _L,X and P _L,O are the permeability coefficients for the lipoidal pathway when the solvent is enhancer/PBS and PBS, respectively, and S _Xand S _Oare the permeant solubilities in enhancer/PBS and in PBS, respectively. The solubility ratio corrects for any activity coefficient differences between the activity coefficient in PBS and that in the enhancer solution. Use of the solubility ratio assumes that Henry’s law is obeyed for the permeant in both PBS and enhancer solutions (Kim et al. 1992).

7.2.2.4 Permeant Solubility Determination

The solubilities of the steroidal permeants in PBS and the enhancer solutions were determined by adding excess crystals of the permeant into the enhancer solution in Pyrex culture tubes. The drug suspension was shaken for 72 h at 37 °C. The culture tubes were then centrifuged for 15 min at 3500 rpm, and the clear supernatants were analyzed for permeant concentrations with HPLC.

7.2.2.5 Determination of Partition Coefficient in Bulk Organic Solvent/PBS Systems

Organic solvent/PBS partition coefficients were obtained at the aqueous enhancer concentrations corresponding to E = 10 and at one tenth of the E = 10 concentration, the latter to test whether Henry’s law is obeyed in the two liquid phases. The two-phase systems were maintained at 37 °C for 72 h. Both the organic and aqueous phases were centrifuged, and aliquots were carefully withdrawn from both phases and appropriately diluted for subsequent analysis using HPLC or GC.

7.2.3 Partition Experiments

7.2.3.1 n-Heptane Treatment and SC preparation

Before SC preparation, HMS was rinsed with heptane for 3 × 10 s to remove the SC surface lipids. This rinsing protocol (the number of rinses and the rinse time) was shown to remove approximately 20 % of the SC lipids but did not disrupt the SC barrier (He et al. 2003). Similar treatments with nonpolar organic solvent were also shown to remove skin surface lipids (e.g., Abrams et al. 1993; Nicolaides 1974). SC was then prepared according to the method described by Kligman and Christophers (1963) and Yoneto et al. (1998). Briefly, the skin was placed, dermis side down, on a filter paper (quantitative filter paper No. 1, Whatman^®) mounted on a Petri dish. The Petri dish was filled with 0.2 % trypsin in PBS solution up to the surface of SC. The Petri dish was covered and maintained at 37 °C for 16 h. When the skin membrane was placed in distilled water after the trypsin treatment, the dermis and viable epidermal layers would separate and fall away from the SC. The SC was then rinsed with distilled water several times and swabbed with Kimwipe^® tissue paper to remove excess water. Then, the SC was placed on aluminum foil and dried at room temperature. After drying, the SC was kept in a freezer for later use.

7.2.3.2 HMS SC Delipidization

Heptane-treated HMS SC samples were prepared as described in the previous section. The delipidized HMS SC was prepared according to the method described previously (Yoneto et al. 1998). Briefly, dried n-heptane-treated SC samples (about 1–2 mg) were weighed and transferred into 5 mL CHCl₃/MeOH (2:1) mixture and equilibrated for 48 h at room temperature. The residue of SC was then rinsed several times with fresh CHCl₃/MeOH (2:1) mixture and dried under room temperature for 24 h. The dried residue was carefully weighed and used for the partition experiments.

7.2.3.3 Partition Experiments with Heptane-Treated and Delipidized HMS SC

Partition experiments were carried out to determine the uptake amounts of the chemical permeation enhancer and of probe permeant ES into n-heptane-treated or delipidized HMS SC. Two different partition experimental setups were used in our laboratory. The old setup used a Franz diffusion cell (Yoneto et al. 1998) and would not be discussed here. The following is a brief description of the other method (Chantasart et al. 2004). Heptane-treated SC (about 1–2 mg) or delipidized SC sample was carefully weighed and equilibrated in about 20 mL of enhancer solution (E = 10 concentration) containing trace amounts of radiolabeled ES (³H-ES) in a screw-capped glass vial. The vial was sealed with parafilm to prevent enhancer solution evaporation and put in a water bath with shaking at 37 ± 0.1 °C for 12 h. The 12-h incubation period was chosen because preliminary studies showed that equilibrium of enhancer and ³H-ES with the SC sample took place in less than 12 h and that a longer incubation period might result in too fragile a membrane sample for the partitioning experiments. After 12 h, the SC sample was taken out from the solution by tweezers and blotted by Kimwipe^® tissue paper. The enhancer and ES concentrations of the solution in the screw-capped glass vial were checked. The wet SC sample was carefully weighed in a snap-capped glass bottle. Then, 5 mL of 100 % ethanol was added into the bottle to extract the enhancer and ES from the sample for 48 h at room temperature with occasional gentle agitation. The extracted solution was then transferred to a screw-capped Pyrex test tube. The test tube was centrifuged at 3500 rpm for 15 min. The supernatant was analyzed for the enhancer by GC or HPLC and for ES by a scintillation counter.

The uptake amount of enhancer in the heptane-treated SC or delipidized SC was calculated as follows:

${A}_{{}_{\mathrm{corrected}, i}}=\frac{A_{\mathrm{extracted},\i}}{W_{\mathrm{dry}}}-\left({W}_{\mathrm{wet}}-{W}_{\mathrm{dry}}\right)\frac{Ci}{W_{\mathrm{dry}}}$

(7.7)

where A _{extracted, i}is the amount enhancer extracted from heptane-treated or delipidized HMS SC, W _dry is the dried heptane-treated or delipidized SC weight, and the subscript i represents the enhancer. A correction for the enhancer in the aqueous compartment(s) of the SC was calculated according to the wet weight of SC (W _wet) and the concentration of the enhancer in aqueous bulk phase (C _i). The partition coefficient of ES (K _ES) for partitioning from the aqueous phase into n-heptane-treated SC or delipidized SC was calculated as follows:

${K}_{\mathrm{ES}}=\frac{\left[{A}_{\mathrm{extracted}}^{\prime }-\left({W}_{\mathrm{wet}}-{W}_{\mathrm{dry}}\right){C}_i^{\prime}\right]/{W}_{\mathrm{dry}}}{C^{\prime }{}_i{}}\frac{S_{\mathrm{X}}^{\prime }}{S_{\mathrm{O}}^{\prime }}$

(7.8)

where A′ _extracted is the amount of extracted ³H-ES, C′ _iis the concentration of ³H-ES in aqueous bulk phase, and S′ _X and S′ _O are the solubilities of ES in enhancer solution and in PBS, respectively. The solubility ratio corrects for any activity coefficient differences between the activity coefficient of ES in PBS and that in the enhancer solution.

7.2.3.4 Permeant Partitioning into the Transport Rate-Limiting Domain and Equilibrium Permeant Partitioning into the Stratum Corneum Intercellular Lipids

An important question raised in the above transport and equilibrium partition studies was: is the equilibrium partition enhancement data of ES a direct correlate of the partition enhancement of ES in SC permeation? To address this question, the partition enhancement in SC permeation was determined in skin transport experiments using a nonsteady state transport analysis (He et al. 2005). However, a direct comparison of the partition enhancement data obtained in transport experiments and those data obtained in equilibrium partitioning experiments of ES was not practical due to the D/E layer being a significant barrier for ES permeation across HMS. Furthermore, significant ES metabolism was observed in ES transdermal permeation. Because of these difficulties, nonsteady state ES transport analysis was complicated, and it was decided to employ CS as the surrogate permeant for ES in the following study. The strategy here was to examine the relationship between the transport partitioning enhancement of CS and the equilibrium partitioning enhancement of ES, with the assumption that ES and CS should likely behave similarly. This assumption was considered to be reasonable because previous studies had shown similar permeability coefficient enhancement effects of chemical enhancers with ES and CS for permeation across the lipoidal pathway of HMS SC (Yoneto et al. 1995).

The skin transport model (He et al. 2005) is a two-layer numerical transport simulation with a least squares-fitting software Scientist (MicroMath, Salt Lake City, UT). This model assumes that both SC and D/E are homogenous and divides the SC and D/E into a sufficient number of layers characterized by partition, diffusion, and dimension parameters. The permeant concentration in the donor chamber was assumed constant, which was true in all transport experiments carried out in the study. The receiver chamber concentration was kept at sink conditions. The transport data of full-thickness HMS were analyzed using the model to obtain the partition coefficient (K _SC) and diffusion coefficient (D _SC) of SC. The reduced parameters K _SC′ and D _SC′ of SC were then calculated:

${D}_{\mathrm{S}{\mathrm{C}}^{\prime }}={D}_{\mathrm{S}\mathrm{C}}/{L}^2$

(7.9)

${K}_{S{ C}^{\prime }}={K}_{S C} L$

(7.10)

where L is the effective path length across SC. These reduced parameters K _SC ′ and D _SC ′ were defined (Okamoto et al. 1988) to avoid the difficulty and uncertainty in assigning the L value and to minimize the number of parameters for least square fitting in model analyses of the experimental transport data. The enhancement of K _SC ′ and D _SC ′ (E _{K, SC} and E _{D, SC}, respectively) was calculated by dividing the K _SC ′ and D _SC ′ parameters obtained with the enhancers at E = 10 by those with PBS control.

7.3 Results and Discussion

7.3.1 Isoenhancement Concentrations and Enhancer Effects

Figure 7.2 shows a representative plot of enhancement factor vs. aqueous enhancer concentration for ES, CS, and HC permeation across the SC lipoidal pathway with 1-butyl-2-pyrrolidone, 1-hexyl-2-pyrrolidone, and 1-octyl-2-pyrrolidone as permeation enhancers (Yoneto et al. 1995). Similar enhancement factor vs. aqueous enhancer concentration plots were observed for the enhancers studied. The enhancement factor profiles at increasing aqueous enhancer concentrations are essentially the same for the steroidal permeants of different lipophilicity, suggesting the same mechanism of permeation enhancement for these steroidal permeants.

Fig. 7.2

Transport enhancement factors of estradiol (ES), corticosterone (CS), and hydrocortisone (HC) across the SC lipoidal pathway in the presence of 1-butyl-2-pyrrolidone (BP), 1-hexyl-2-pyrrolidone (HP), and 1-octyl-2-pyrrolidone (OP). The transport enhancement factors were calculated using Eq. (7.6)

7.3.2 Effects of Alkyl Chain Length

The isoenhancement concentrations at E = 10 for more than 20 different enhancers are presented in Fig. 7.3 (Warner et al. 2003); isoenhancement concentration is defined as the aqueous concentrations of enhancers to induce the same enhancement factor and in this case E = 10. These isoenhancement concentrations were interpolated from the E vs. aqueous enhancer concentration plots similar to those in Fig. 7.2. Figure 7.3 shows the relationship between the E = 10 enhancer concentration and the carbon number of the enhancer n-alkyl group (at constant permeant thermodynamic activity). The major conclusion deduced from the data in Fig. 7.3 is a slope of around −0.55 found for each enhancer series (enhancers have the same polar head functional group but different alkyl chain length) in the figure. The value of −0.55 translates into an around 3.5-fold increase in potency per methylene group for the enhancers. In other words, the aqueous concentration required to induce E = 10 increases 3.5-fold when the alkyl chain length of the enhancer decreases by one methylene group. The constant slope of −0.55 for the different enhancer series suggests a hydrophobic effect involving the transfer of the methylene group from the aqueous phase to a relatively nonpolar organic phase (e.g., Tanford 1980).

Fig. 7.3

Relationships between the aqueous E = 10 isoenhancement concentrations of the enhancers and the carbon number of the enhancer alkyl chain. Each data point represents the average value without showing the standard deviation because the error bar generally lies within the symbol in the plot. Enhancer abbreviations are provided in Fig. 7.1

The results of the equilibrium partition experiments with the enhancers conducted to determine the amount of enhancers in the SC intercellular lipids under the isoenhancement E = 10 conditions (He et al. 2003, 2004) are shown in Fig. 7.4. Note that the scale of the y-axis in Fig. 7.4 is the same as that in Fig. 7.3. The data in Fig. 7.4 suggest that there was little effect of the enhancer alkyl chain length upon the enhancer potency based on the concentrations of the enhancers in the intercellular lipid lamellae (relative to that based on the E = 10 aqueous enhancer concentrations in Fig. 7.3), thus suggesting that the intrinsic potency of the enhancers at their site of action, generally believed to be the SC intercellular lipids, is relatively independent of their alkyl group chain length and lipophilicity.