Skin Permeation of Testosterone from Viscoelastic Lecithin Reverse Wormlike Micellar Solution

Abstract

We evaluated testosterone-containing lecithin reverse wormlike micelles (reverse worms) composed of a polar substance/lecithin/isopropyl myristate for transdermal application. Water, D-ribose, or tetraglycerol were used as the polar substance and were key ingredients for forming the reverse worms. Using the reverse worms, 1 wt% of testosterone could be stably solubilized. When using D-ribose as polar substance, the maximum zero-shear viscosity of the reverse worms solution was higher than that of systems using water or tetraglycerol as the polar substance. The mechanism of skin permeation of testosterone from reverse worms solution was elucidated using skin permeation experiments with hairless mouse skin. When the structure of the reverse worms transitioned to lamellar liquid crystals at the skin/formulation interface, testosterone became supersaturated in the formulations. The structural transition occurred in systems using water or D-ribose as the polar substance, increasing the flux of testosterone. The flux of testosterone from reverse worms solution thus depends on the type of polar substance used.

Recent advances in drug dosage forms have led to improved quality of life for patients. The transdermal dosage form, in particular, represents one of the most important drug delivery methods. Transdermal dosage forms enable avoidance of the hepatic first-pass effect, which can be problematic with oral administration. Another advantage of transdermal dosage forms is that they are easy to apply and remove from the skin. Based on these advantages, transdermal dosage forms have been adopted for a variety of medications.

We investigated the possibility of using lecithin reverse wormlike micelle (called reverse worms) comprised of a polar substance/lecithin/oil as the vehicle for transdermal therapeutic application of testosterone (TES). Lecithin is an amphiphilic molecule composed of both hydrophilic and hydrophobic groups. In general, lecithin forms spherical or ellipsoidal reverse micelles when added alone to oil. When trace amounts of a polar substance (e.g., water)¹⁾ are added to this solution, the polar substance can attach to the phosphate groups of the lecithin molecules via hydrogen bonding, widening the gap between the head groups of neighboring lecithin molecules. Thus, the value of the critical packing parameter decreases and the interface curvature of the molecular assembly subsequently induces the formation of reverse worms. As a result, phase transition from reverse spherical micelles to reverse worms takes place. These reverse worms become tangled in the oil and form a three-dimensional (3D) network throughout the solution, turning the solution into an gel-like solution (also called a lecithin organogel).^1–6) In this reverse micellar system the polar substance is the key ingredient for the formation of reverse worms. We have also identified other key ingredients that can be used as substitutes for water in the preparation of reverse worms in n-decane, such as urea,⁷⁾ sucrose fatty acid esters,⁸⁾ D-ribose,⁹⁾ 2-deoxy-D-ribose,⁹⁾ polyglycerols,¹⁰⁾ ascorbic acid,¹¹⁾ and multivalent carboxylic acids.¹²⁾

Lecithin is not only biocompatible, it also promotes the percutaneous absorption of various drugs.^13–17) Reverse worms prepared using biocompatible oils in combination with lecithin and a polar substance permit the percutaneous absorption of preparations with minimal skin irritation. Such reverse worms can be used to deliver poorly soluble drugs composed of micellar structures. Despite these advantages, however, a few reports have been published regarding the use of reverse worms systems for percutaneous drug administration.¹⁷⁾

We previously studied the physicochemical properties of reverse worms prepared using n-decane. However, n-decane is not suitable for external dosage forms. Thus, we used isopropyl myristate (IPM) as the oil in this study. IPM reportedly acts as a percutaneous absorption accelerator.¹⁸⁾ With that in mind, we investigated the properties of a polar substance/lecithin/IPM/drug quaternary system. TES was used as model drug for the experiments in this study. The solubilizing ability of reverse worms would be effective to solubilize poorly soluble drug, TES. Also transdermal therapeutic application is suitable for TES to avoid first-pass metabolize in liver and to keep constant blood level. Water, D-ribose, and tetraglycerol (4PGL) were chosen as polar substances. Water is the standard polar substance for forming highly viscoelastic reverse worms solution, and the use of reverse worms solution for transdermal delivery has been reported. In polar substance/lecithin/n-decane systems, D-ribose can induce the formation of highly viscoelastic reverse worms solution.⁹⁾ In contrast, 4PGL can induce the formation of reverse worms solution over a wide concentration range.¹⁰⁾

In this study, we examined the physicochemical properties of reverse worms solution containing TES and investigated the skin permeation by in vitro experiments utilizing hairless mouse skin. The mechanism of skin permeation of TES from reverse worms solution was also elucidated.

MATERIALS AND METHODS

Materials

Soybean lecithin (PHOSPHOLIPON90G; PC content=min 95%) was purchased from H. Holstein Gmbh & Co. (Hamburg, Germany). D(−)-Ribose, TES, IPM, and Dulbecco’s phosphate-buffered saline (PBS) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and 4PGL was donated by Daicel Co., Ltd. (Tokyo, Japan). Water was purchased from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Acetonitrile and methanol were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

Sample Preparation

For solid polar substances, the required amount of lecithin, polar substance (D-ribose), and TES were dissolved in methanol in a vial, and then the solvent was completely removed in a desiccator equipped with a vacuum pump. Following addition of IPM, the solution was mixed using a magnetic stirrer. The resulting solutions were stored at 25°C for several days to allow for equilibration.

For liquid polar substances, the required amount of lecithin, polar substance (water or 4PGL), TES, and IPM were mixed in a vial using a magnetic stirrer. The resulting solutions were stored at 25°C for several days to allow for equilibration.

Construction of Phase Diagrams

Phase diagrams were constructed based on visual observation through crossed polarizers and by small-angle X-ray scattering (SAXS) analysis. SAXS was performed using a Nano-STAR instrument (Bruker AXS Inc., Madison, WI, U.S.A.) equipped with a CuKα radiation source operated at 45 kV/120 mA. All measurements were performed at 25°C.

Rheological Measurements

Steady and dynamic rheological measurements were performed using a stress-controlled rheometer (HAAKE RS600, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). A double-cone geometry (60-mm diameter, 1° cone angle) or a cone-plate geometry (35- or 60-mm diameter, 1° cone angle) was used. The temperature was maintained at 32°C using a Peltier-based temperature-control device. For steady-flow viscosity measurements, the samples were subjected to the desired shear stress for a time sufficient to achieve a steady state. Dynamic rheological measurements were performed using the strain (γ) value from the linear viscoelastic region. A solvent trap was used to prevent evaporation.

Skin Permeation Experiments

Frozen samples of excised skin of hairless mice (Laboskin^®, Hos: HR-1 Male, 7 weeks old, Hoshino Laboratory Animals Inc., Ibaraki, Japan) were used in permeation experiments. Skin was thawed and placed on a Franz-type diffusion cell (Hanson Research Co., Chatsworth, CA, U.S.A.) with a receptor volume ranging from 7.0 to 7.3 mL and a diffusional area of 1.74 cm². PBS was added to the receptor cell, which was maintained at 32°C. The receptor cell was stirred using a magnetic stirrer (550 rpm). One gram of samples containing TES (10 mg/g) were placed in the donor compartment on the skin. A 0.5 mL sample of the receptor fluid was collected at predetermined times and immediately replaced with fresh PBS. The concentration of TES in the removed receptor fluid was determined using HPLC.

HPLC Analysis of TES

The HPLC system consisted of a UV detector (UV-2075, JASCO Co., Tokyo, Japan), a Chromatopac data processor (C-R8A, Shimadzu Co., Kyoto, Japan), and an HPLC pump (PU-2080, JASCO Co.). An L-column2 ODS (Chemical Evaluation and Research Institute, Tokyo, Japan) was used, and the mobile phase consisted of a degassed mixture of acetonitrile and water (1 : 1). The flow rate was set at 1.0 mL/min, and the detection wavelength was set at 241 nm.

Polarizing Microscopy

Polarizing microscopy was performed using an ECLIPSE 600W POL polarizing microscope (Nikon Co., Tokyo, Japan).

Data Analysis

All data are expressed as the mean±standard deviation (S.D.). The significance of differences was determined using ANOVA followed by Fisher’s protected least significant difference test. A p value <0.05 was considered indicative of statistical significance in all cases.

RESULTS AND DISCUSSION

Physicochemical Properties of TES-Containing Highly Viscoelastic Reverse Worms Solution

Figure 1 shows photographs of D-ribose/lecithin/IPM/TES solutions at varying D-ribose concentrations, where the lecithin and TES concentrations were fixed at 30 and 1 wt%, respectively. At low D-ribose concentrations, the solution exhibited low viscosity (Fig. 1a). However, with increasing D-ribose concentration, the viscosity increased and the sample flowed very slowly in the inverted vial (Fig. 1b). Finally, at a certain D-ribose concentration, the solution exhibited gel-like behavior (Fig. 1c).

Fig. 1. Photographs of TES-Containing Reverse Worms Prepared Using D-Ribose as the Polar Substance (25°C)

(a) D-Ribose/lecithin/IPM/TES (3.75 : 30.0 : 65.25 : 1 [wt%]); (b) D-Ribose/lecithin/IPM/TES (4.85 : 30.0 : 64.15 : 1 [wt%]); (c) D-Ribose/lecithin/IPM/TES (6.18 : 30.0 : 62.82 : 1 [wt%]).

SAXS was used to determine the structure of the reverse worms. Samples used for SAXS measurements were diluted 15-fold to eliminate structure–factor effects. Figure 2 shows the SAXS scattering intensity (q=[4π/λ] sin θ, where λ represents the X-ray wavelength and 2θ represents the scattering angle) for the D-ribose/lecithin/IPM/TES (0.41 : 2.0 : 97.52 : 0.067 [wt%]) system. In this profile, the slope of the double logarithmic plot in the low-q region was −1, which was indicative of cylindrical particles (i.e., the presence of reverse worms). The appearance changes indicating phase transition from reverse spherical micelles to reverse worms are shown in Fig. 1. If the quantity and length of reverse worms are sufficient, the solution changes into a transparent gel-like solution, a process we have described elsewhere.^7,8,10)

Fig. 2. SAXS Intensity [I(q)] as a Function of the Scattering Vector (q) for the D-Ribose/Lecithin/IPM/TES (0.41 : 2.0 : 97.52 : 0.067 [wt%]) System

Next, to confirm the formation of reverse worms exhibiting high viscoelasticity, we constructed partial phase diagrams for polar substance/lecithin/IPM/TES systems using water, D-ribose, or 4PGL as the polar substance (Fig. 3). In all cases, the TES concentration was fixed at 1 wt%. The phase diagrams were constructed based on visual observation through crossed polarizers or via SAXS analysis. In all systems, reverse micelles (Om) formed upon the addition of a small amount of polar substance, and the presence of a highly viscoelastic region (shown by shading) was confirmed within the Om regions. Of note, in this study, we defined regions with a η₀ value of ≥100 Pa·s as highly viscoelastic. The highly viscoelastic region contained reverse worms of sufficient length and quantity to form 3D network structures. As such, in the 2-phase region around the lecithin-IPM axis, the lecithin does not dissolve in the oil, and the boundary between the 2-phase and reverse micellar regions is the Krafft points (melting point of solvated solid lecithin). That is, the boundary indicates a composition of polar substance/lecithin/IPM/TES with a Krafft point of 25°C. In the 2-phase region in which the polar substance concentration was high, phase separation occurred as a result of shrinkage of the reverse worms following addition of excess polar substance or the formation of lamellar liquid crystals at high lecithin concentrations. The phase diagrams indicate that 1 wt% of TES can be stably solubilized using reverse micelles in the reverse micellar region. Of note, the 4PGL/lecithin/IPM/TES system exhibited the largest reverse micellar region, whereas the D-ribose/lecithin/IPM/TES system exhibited the largest highly viscoelastic region. These differences originate from differences in the polarity, size, and structure of the polar substances. Furthermore, we found that the highly viscoelastic regions of polar substance/lecithin/IPM/TES quaternary systems were smaller than those of polar substance/lecithin/IPM ternary systems (data not shown). These results suggest that the critical packing parameter (CPP) value of the molecular assembly changes slightly when TES is solubilized in reverse worms. Because of this change, the length and quantity of the reverse worms become insufficient for the formation of the 3D network structure, and the highly viscoelastic region becomes smaller.

Fig. 3. Partial Phase Diagrams for Polar Substance/Lecithin/IPM/TES Systems in the Dilute Region at 25°C

The polar substances were water (upper), D-ribose (middle), and 4PGL (bottom). The TES concentration was fixed at 1 wt%. The notation ‘Om’ indicated the reverse micellar phase region. The highly viscoelastic region within the Om phase is shown by shading. Black dot and black star were used in skin permeation study.

Figure 4a shows the results of steady-flow viscosity measurements for the D-ribose/lecithin/IPM/TES system. All reverse worms solutions exhibited Newtonian flow behavior in the low shear-rate range, as demonstrated by a nearly constant viscosity. This indicates that the 3D network structure formed by the reverse worms is not broken in this shear-rate range. Micelle formation is actually an equilibrium process in which micelles are in equilibrium with unassociated surfactant molecules (monomers). The 3D network structure of the reverse worms formed in this system is not static like a synthetic polymer but instead is a transient network in which collapse and formation are repeated. Therefore, if the reformation rate of the reverse worms is sufficiently higher than the shear rate, the reverse worms solution exhibits Newtonian flow because the network structure does not break. At higher shear rates, however, the reverse worms solution exhibited non-Newtonian flow behavior. Similar viscosity behaviors were also observed for water/lecithin/IPM/TES, 4PGL/lecithin/IPM/TES systems and ternary systems without TES. The η₀ value, considered in more detail below, was obtained by extrapolation of the steady flow viscosity curve to a shear rate of zero.

Fig. 4. (a) Steady Shear-Rate Viscosity (η) Curves for D-Ribose/Lecithin/IPM/TES Systems (32°C); (b) Relationship between Zero-Shear Viscosity (η₀) and Polar Substance Concentration (32°C)

The lecithin and TES concentrations were fixed at 30 and 1 wt%, respectively.

Figure 4b shows the relationship between zero-shear viscosity (η₀) and polar substance concentration in reverse worms solution. The η₀ values of all systems monotonically increased until phase separation or cloudiness occurred. These results indicate that the polar substances examined (D-ribose, water, and 4PGL) induce the formation of reverse worms with sufficient length for entanglement. Importantly, these results also indicate that the viscosity of the reverse worms solution can be adjusted easily by adjusting the amount of polar substance added. However, the range of viscosity over which the solution can be adjusted depends on the type of polar substance used. The zero-shear viscosity of the D-ribose/lecithin/IPM/TES system can reach about 400 Pa·s, but that of the 4PGL/lecithin/IPM/TES system can reach only about 60 Pa·s, and that of the water/lecithin/IPM/TES system reaches only about 10 Pa·s. It is noteworthy that the rate of the increase in viscosity also depends on the type of polar substance. For example, in the case of the 4PGL/lecithin/IPM/TES system, it is necessary to add 10.2 wt% of 4PGL to adjust the zero-shear viscosity to 10 Pa·s. In the case of the water/lecithin/IPM/TES system, in contrast, the addition of only 3.4 wt% of water is necessary to achieve the same viscosity. Based on the results described above, D-ribose should be chosen as the polar substance in order to prepare high-viscosity reverse worms solution.

Next, dynamic viscoelasticity measurements (variation in the storage modulus [G′] and loss modulus [G″] as a function of frequency [ω]) were carried out in order to characterize the viscoelasticity of TES-containing reverse worms solution. Here, G′ and G″ represent elasticity and viscosity, respectively. Figure 5 shows the results of dynamic viscoelasticity measurements for D-ribose/lecithin/IPM/TES systems, in which the lecithin and TES concentrations were fixed at 30 and 1 wt%, respectively. The reverse micellar solutions with a low zero-shear viscosity (1 and 10 Pa·s) exhibited a viscous response, with G″ exceeding G′ over the entire ω range. By comparison, the reverse micellar solution with a high zero-shear viscosity (100 Pa·s) exhibited a viscoelastic response, with the curves for G′ and G″ intersecting at 22 rad/s. At high frequencies, the elasticity component was predominant (G′>G″), whereas at low frequencies, the viscosity component was predominant (G″>G′). These results indicate that a reverse worms solution with a high zero-shear viscosity (100 Pa·s) behaves as a viscoelastic body. These viscoelastic behaviors can be observed in systems using water or 4PGL as the polar substance and are generally characteristic of reverse worms.

Fig. 5. Variations in G′ and G″ as a Function of ω at Different D-Ribose Concentrations in D-Ribose/Lecithin/IPM/TES Systems

The lecithin and TES concentrations were fixed at 30 and 1 wt%, respectively.

Skin Permeation of TES from Highly Viscoelastic Reverse Worms Solution

To investigate the characteristics of TES-containing reverse worms solution for use as percutaneous absorption preparations, we performed in vitro skin permeation experiments using hairless mouse skin. Table 1 shows the compositions and abbreviations of the formulations used. To maintain the viscosity of reverse worms solution, the lecithin and TES concentrations were fixed at 30 and 1 wt%, respectively. If the lecithin concentration is low or TES concentration is high, the viscosity of formulation decreases. Figure 6 shows the typical TES permeation profiles for the controls and W1. In each system, TES permeated the skin in a steady-state fashion. The cumulative amount of TES that had permeated the skin from W1 by 10 h after application was about double that of controls 1 and 2. The flux of TES through the skin from each sample was determined from the slope of the linear portion of the plot. The X-axis intercept was taken as the lag time.

Fig. 6. Permeation Profiles of TES from Control 1, Control 2, and W1 Reverse Worms at 32°C

Each point represents the mean±S.D. of at least three experiments.

Table 1. Compositions of the Formulations (wt%)

	Control		Reverse worms
	1	2	W1	W2	W3	R1	R2	R3	P1	P2	P3
Lecithin		30	30	30	30	30	30	30	30	30	30
TES	1	1	1	1	1	1	1	1	1	1	1
IPM	99.0	69.0	65.6	67.0	67.5
Water			3.4	2.0	1.5	62.82	64.15	65.25
D-Ribose						6.18	4.85	3.75	55.6	58.8	61.5
4PGL									13.4	10.2	7.5

Compositions of R1, R2, and R3 correspond to photos (c), (b), and (a) in Fig. 1, respectively.

The flux of TES from reverse worms and controls was compared (Fig. 7a). The flux of TES depended on the polar substance used, with a trend in the flux value as follows: W1>R1>P1. Because the viscosities of the samples in this experiment differed, the effect of viscosity on the rate of skin permeation was investigated. Table 2 shows the flux of TES from reverse worms solution of different viscosity. Viscosity did not have a substantial impact on TES flux in any of the systems. These data clearly demonstrate that the viscosity of the vehicle has no effect on permeation of the skin of TES. These results also clearly show that the concentration of polar substance, which determines the viscosity of the reverse worms solution, does not affect permeation of the skin of TES.

Fig. 7. Comparison of (a) Flux and (b) Lag Time between Control 1, Control 2, and Viscoelastic Reverse Worms

Each point represents the mean±S.D. of at least three experiments. * Significant difference relative to control 1 (p<0.05).

Table 2. Flux and Lag Time of TES from Reverse Worms at Various Viscosities (32°C)

Polar substance		η0 (Pa·s)	State	Flux (µg/cm2/h)	Lag time (h)
Water	W1	10	Viscoelastic	3.98±0.21	1.06±0.08
	W2	1	Viscous	3.96±0.39	0.96±0.08
	W3	0.3	Viscous	3.94±0.32	1.17±0.34
D-Ribose	R1	100	Viscoelastic	2.69±0.25	0.77±0.13
	R2	10	Viscous	2.91±0.23	1.04±0.08
	R3	1	Viscous	3.21±0.49	1.20±0.53
4PGL	P1	50	Viscoelastic	2.35±0.12	1.02±0.09
	P2	10	Viscous	2.27±0.23	1.24±0.09
	P3	1	Viscous	2.39±0.19	0.74±0.15

Each point represents the mean±S.D. of at least three experiments.

In general, maximum flux is achieved when a drug is applied in suspension form¹⁹⁾; therefore, if the formulation has no effect on permeation of the skin, the flux values for all formulations would be expected to be similar to that of control 1 (the solubility of TES in IPM is 29 µg/mL). However, the flux values from reverse worms solution using water or D-ribose as the polar substance were significantly higher than that of control 1. Figure 7b shows the lag time of TES for each formulation. W1, R1, and P1 each exhibited a lag time of about 1 h, significantly longer than that of control 1, indicating that the increment of the diffusion rate in the skin cannot explain TES permeation.

Willimann et al. reported that reverse worms solution facilitate drug permeation, but they hypothesized that this is primarily due to interaction between the phospholipid (lecithin) and the stratum corneum.¹⁶⁾ The flux, however, tended to differ among the polar substances in spite of the same lecithin concentrations in our study. Therefore, we must consider the possibility that another factor affects drug flux. As such, we examined the state of the formulations on the skin after drug permeation. In W1 and R1 on the skin were locally turbid at the interface with the skin. In contrast, P1 maintained transparency. We hypothesized that water in the skin was absorbed by the formulations at the interface and the CPP of the molecular assembly changed. To confirm this structural transition, the state of the formulations after skin permeation was observed using polarizing microscopy. Figure 8 shows polarization micrographs of W1, R1, and P1 and controls before and after the skin permeation experiment. In control 1, a suspension, TES crystals were observed both before and after the experiment. TES crystals were observed in control 2 before the skin permeation experiment because the solubility of TES in lecithin/IPM is <10 mg/mL. After the experiment, however, polarization of the lamellar structure was observed, but no TES crystals were apparent. We hypothesized that control 2 was affected by water from the skin and that phase transition from suspension to lamellar liquid crystal via the reverse micellar phase took place in the formulation on the hairless mouse skin. This phenomenon could be explained by the phase diagram shown in Fig. 3, in which the composition of control 2 is marked by a black star. Absorbing water from skin, this composition moves in the direction of the arrow toward the composition of reverse micellar region before assuming a lamellar liquid crystal composition. The process involves 2 steps, and after this 2-step structural transition, permeation of TES was enhanced. Thus, the amount of permeated at 10 h after application was not high and the lag time was longer than other formulation. In the case of control 2, the lag time was increased further because of this 2-step structural transition. No polarizing was observed in the pre-experiment micrographs of W1, R1, and P1 in Fig. 8 as these were reverse worms solutions which were transparent and optical isotropic. In the micrographs of W1 and R1 after the experiment, however, polarization characteristic of lamellar structures was observed, similar to the case of control 2. No polarization was observed for P1 after the experiment. In the case of W1, when water was absorbed by the reverse worms solution (indicated by a black dot on the phase diagram in Fig. 3 for the system using water), the water/lecithin composition moved in the direction of the arrow, assuming that of a lamellar liquid crystal. In the case of R1, the explanation is difficult because water from the skin is the second polar substance for this system.

Fig. 8. Polarization Micrographs of Control 1, Control 2, and Viscoelastic Reverse Worms at 25°C

Images were taken before and after skin permeation experiments.

To reproduce this phenomenon, water was added to 1 g of W1 and R1 reverse worms, which were then stirred. The W1 and R1 reverse worms became turbid with 1 and 30 µL of water, respectively. These observations indicate that the structural transition from reverse worms to lamellar liquid crystals occurs when water is absorbed. A lower quantity of water is thus needed to induce the structural transition in formulations using water than in formulations using D-ribose.

There was possibility that the lamellar liquid crystals have different capacity for solubilizing TES from the reverse worms. No crystals of TES were observed immediately after the skin permeation experiment in both W1 and R1. TES crystals were observed after overnight storage in reverse worms samples to which water had been added. This indicates that TES in lamellar liquid crystals reduced TES solubility and is in a supersaturation state, having high energy during the skin permeation experiment. Therefore, the flux of TES from control 2, W1, and R1, in which lamellar liquid crystals formed, was higher than the flux from control 1. This supersaturation state of TES is kept for sufficient period in association with the viscosity of the formulation and the presence of large molecules such as lecithin.

The reason the structural transition did not occur in the system using 4PGL could be due to the properties of 4PGL. Previous observations indicated that 1 g of P1 needs 200 µL of water to change lamellar liquid crystals. 4PGL, which has many hydroxyl groups, can absorb a considerable amount of water, and the highly viscoelastic reverse worms solution using 4PGL are thus minimally affected by the presence of water in the skin.

We also examined the effect of the polarity of the polar substance on the TES permeation rate and destabilization (supersaturation) of TES at the skin/formulation interface. The polarity of polar substances examined in this study is in the order water>D-ribose>4PGL. The polarity was calculated from organic conceptual diagram.^20,21) As TES is more stable in lamellar liquid crystals containing D-ribose than in lamellar liquid crystals containing water, TES is more readily distributed to the skin and exhibits a higher flux in micellar solutions using water.

From these results, we suggest that in TES-containing reverse worms solution, a structural transition from reverse worms to lamellar structures occurs, resulting in TES becoming supersaturated, and leading to an increase in drug permeation. Furthermore, whether this structural transition occurs depends on the polar substance used. Because transepidermal water loss between hairless mouse skin (9.7±1.5 g/m²/h)²²⁾ and human skin (from 12.0±3.0 to 25.9±6.4 g/m²/h depend on anatomic site)²³⁾ are similar level, this structural transition will be occurred on human skin.

CONCLUSION

Reverse worms prepared using IPM as the oil, and water, D-ribose, or 4PGL as the polar substance, can contain 1 wt% TES and form a transparent highly viscoelastic solution. As the viscosity of the solution depends on the concentration of the polar substance used, this viscosity can be adjusted easily.

Skin permeation of TES was enhanced using revers worms with water or D-ribose. The mechanism of enhancement is the structural transition from reverse worms to lamellar liquid crystals at the skin/formulation interface, causing TES to enter a supersaturation state in the formulation. Our results indicate that the polar substance should be chosen based upon the specific intended purpose for the reverse worms for transdermal drug delivery.

Acknowledgments

Thanks are due to Ms. Natsumi Nishida, Mr. Shinya Morita, Mr. Takahiro Yamada, Ms. Mio Iwasawa, and Ms. Tomoko Takagi for their technical assistance in the experimental work. This work was partially supported by the Japan Science and Technology Agency (JST) A-step feasibility study program, No. AS242Z03620Q.

Conflict of Interest

The authors declare no conflict of interest.

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