Animals

Seven-week-old male Wistar rats (weighing approximately 200 g) were provided by the Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan), and all animal experiments were performed according to the guidelines of Kindai University, the Japanese Pharmacological Society, and NIH. The experimental protocols were approved on April 1, 2019, by Kindai University under the project identification code KAPS-31-011, and the study was carried out in compliance with the ARRIVE guidelines and the AVMA euthanasia guidelines 2020. The rats were housed at 25 °C, and allowed free access to water and a standard CE-2 diet (Clea Japan Inc., Tokyo, Japan).

Chemicals

We used commercially available chemicals of highest purity in this study. Briefly, FEL, l-menthol, cytochalasin D, and isoflurane were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and commercially available FEL stick ointment 3% (CA-FEL ointment) was obtained from Mikasa Seiyaku Co., Ltd. (Tokyo, Japan). Dynasore and rottlerin were provided by Nacalai Tesque (Kyoto, Japan), and carboxypolymethylene (Carbopol® 934) was obtained from Serva (Heidelberg, Germany). Pentobarbital was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Methylcellulose (MC) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan), and 2-hydroxypropyl-β-cyclodextrin (HPβCD) was provided by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Membranes of pore size 20 μm for the drug release study were purchased from GVS Japan (Tokyo, Japan).

Design of carboxypolymethylene gel-based l-menthol-containing FEL nanoparticles

Carboxypolymethylene gel incorporating FEL micro- or nanoparticles was prepared according to our previous reports [17,18,19]. Briefly, FEL was milled in an agate mortar for 1 h, which was subsequently transferred to 2 mL tube filled with 2 mm zirconia beads, and crushed (3,000 rpm, 30 s) at 4 °C using the bead mill method and Bead Smash 12 (Wakenyaku Co. Ltd, Kyoto, Japan). After that, FEL and MC were added to 5% HPβCD solution (in distilled water) and crushed using 0.1 mm zirconia beads and ShakeMaster® NEO (Bio Medical Science, Tokyo, Japan). The bead mill treatments were performed for 180 min at 1,500 rpm, and the milled dispersion was ultrasonicated. These dispersions containing milled FEL were gelled by mixing carboxypolymethylene dissolved in distilled water with 2% l-menthol, and determined as the FEL-NP gel in this study. We prepared the gels incorporating FEL microparticles (FEL-MP gel) by dispersing FEL (original crystal, microparticles), MC, and HPβCD in distilled water, which was gelled by the mixing with carboxypolymethylene dissolved in distilled water with 2% l-menthol. The content of additives, such as l-menthol, were decided according to our previous study [17, 18], and the composition of the FEL-MP and FEL-NP gels was 1.5% FEL, 0.5% MC, 5% HPβCD, and 2% carboxypolymethylene [% (w/w) in distilled water].

Particle characteristics of FEL gel

In this study, the particle size of FEL gel in dispersion containing FEL (0.3 g of FEL gel was diluted with 100 mL of distilled water and stirred for 10 min) was measured using SALD-7100 (Shimadzu Corporation) and NANOSIGHT Software NTA (Quantum Design Japan, Inc.). The measurement conditions for SALD-7100 were a maximum scattered light intensity in the range of 40–60% and a refractive index of 1.60 ± 0.10i. The measurement conditions for NANOSIGHT were a wavelength of 405 nm (Blue), time of 60 s, and viscosity of 1.27 mPa·s. In addition, dispersions containing FEL were set to a scanning probe microscope (SPM-9700; Shimadzu Corp., Kyoto, Japan) to obtain atomic force microscopy (AFM) images represented by the combination of the phase and height images of FEL.

Measurement of FEL concentration

Fifty microliters of samples containing FEL were added to 100 µL of methanol and used to measure the FEL concentration using Shimadzu LC-20AT system equipped with a column oven CTO-20 A (Shimadzu Corp., Kyoto, Japan). The samples (10 µL) were injected using an auto injector SIL-20AC (Shimadzu Corp., Kyoto, Japan). The measurement conditions were as follows: column: Inertsil®ODS-3 (3 μm; GL Science Co. Inc., Tokyo, Japan); column temperature, 35 °C; flow rate, 0.25 mL/min; mobile phase, 0.3% phosphoric acid containing 1% sodium dodecyl sulfate-acetonitrile (70/30 v/v); detection wavelength, 254 nm.

Viscosity of FEL gel

The viscosity of FEL gel was measured using Brookfield digital viscometer with a CPZ-52Z plate (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA). The measurement conditions were as follows: rotational speed, 60 rpm; measurement time, 3.5 min; and temperature, 20 °C.

Drug solubility of FEL gel

The FEL gel dispersions (0.3 g of FEL gel was diluted with 10 mL of distilled water and stirred for 10 min) were centrifuged at 100,000 × g using Beckman Optima™ MAX-XP Ultracentrifuge (Beckman Coulter, Osaka, Japan) to separate the solubilized and non-solubilized FEL. The concentration of soluble FEL (solubility) was determined using HPLC as described above.

Drug release from FEL gel

Drug release from FEL gel was measured according to our previous reports [17,18,19]. Briefly, reservoir chamber (12.2 mL) of Franz diffusion cell was filled with 0.2 mM phosphate buffer (pH 7.2), and membrane of pore size 20 μm was fixed on the cell. After that, 0.3 g of FEL gel (equivalent to 4.5 mg FEL) was spread uniformly over the membrane (A, 2 cm2) and incubated at 37 °C for 24 h. One hundred microliters aliquot samples were withdrawn from the reservoir chamber at 0.5, 1, 2, 3, 6, and 24 h. The FEL concentration and particle size distribution were measured using HPLC and NANOSIGHT, respectively, as described above. The area under the released FEL concentration-time curve (AUCrelease) was calculated using the trapezoidal rule up to the last measurement point (24 h).

Skin penetration of FEL gel

Seventy-two Wistar rats (7-week-old) were divided into nine groups (8 rats/group), and the abdominal skin of the rats was shaved with an electric clipper and razor on the day prior to the experiment. After 24 h, the rats were euthanized by injecting a lethal dose (200 mg/kg) of pentobarbital. The abdominal skin was then removed and placed on a Franz diffusion cell. The in vitro skin penetration of FEL gel was investigated, as described in our previous study [17,18,19]. Briefly, the reservoir chamber (12.2 mL) was filled with 0.2 mM phosphate buffer (pH 7.2), and 0.3 g of FEL gel (equivalent to 4.5 mg FEL) was uniformly spread over the membrane (A, 2 cm2), and incubated at 37 °C for 24 h. Samples (100 µL) were withdrawn from the reservoir chamber at 0.5, 1, 2, 3, 6, and 24 h. The FEL concentration and particle size distribution were measured using HPLC and NANOSIGHT, respectively, as described above. In this study, endocytosis was inhibited by either low temperature incubation or using pharmacological inhibitors. Low temperature inhibition of endocytosis experiment was performed at 4 °C. Different pharmacological inhibitors such as 54 µM nystatin [caveolae-mediated endocytosis (CavME) inhibitor] [20], 40 µM dynasore [clathrin-mediated endocytosis (CME) inhibitor] [21], 2 µM rottlerin [micropinocytosis (MP) inhibitor] [22], and 10 µM cytochalasin D (phagocytosis inhibitor) [20] were used in experiments under thermoregulated (37 °C) conditions. These pharmacological inhibitors were dissolved in 0.5% dimethyl sulfoxide (vehicle) and pretreated for 1 h before the experiment. During the experiment, these inhibitors were added to the reservoir chamber (0.2 mM phosphate buffer). In this study, the area under the penetrated FEL concentration-time curve (AUCpenetration) was calculated using the trapezoidal rule up to the last measurement point (24 h). Moreover, pharmacokinetic parameters [penetration rate, Jc; skin/preparation partition coefficient, Km; skin penetration coefficient, Kp; diffusion constant within the skin, D; lag time, τ; thickness of the skin, δ (0.071 cm); amount of FEL (CC) in the reservoir chamber at time t, Qt; effective area of the skin] were calculated according to Eqs. 1 and 2:

$$}_}} = \frac}_}}}} \cdot \left( } - \tau } \right)}} = \frac} \cdot }_}} \cdot }_}}}} = }_}} \cdot }_}}$$

(1)

$$}\frac}}}}}\tau }}$$

(2)

Percutaneous absorption of FEL gel

Twenty-four Wistar rats (7-week-old) were divided into three groups (8 rats/group), and a cannula filled with 30 µg/mL heparin (silicone tubing; i.d. 0.5 mm, o.d. 1.0 mm) was inserted into the right jugular vein of Wistar rats under isoflurane anesthesia on the day prior to the experiment. The abdominal skin of the rats was shaved with an electric clipper and razor. After 24 h, 0.3 g of FEL ointment and gel (equivalent to 4.5 mg FEL) was spread uniformly over the effective area (2 cm2) of the abdominal skin. Then, 200 µL of blood was collected from the right jugular vein through a cannulation tube at 0.5, 1, 2, 3, 6, and 24 h. The blood samples were centrifuged at 24,000 × g for 20 min at 4 °C, and the plasma obtained was used for the measurement of plasma FEL concentration using HPLC as described above. The area under the FEL concentration-time curve (AUCplasma) was calculated using the trapezoidal rule up to the last measurement point (24 h).

Accumulation of FEL in skin tissue

Seventy-two Wistar rats (7-week-old) were divided into nine groups (8 rats/group). On the day prior to the experiment, the abdominal skin of the rats was shaved with an electric clipper and razor. After 24 h, 0.3 g of FEL ointment and gel (equivalent to 4.5 mg FEL) was spread uniformly over the effective area (2 cm2) of the abdominal skin. At 3, 6, and 24 h after treatment with FEL formulations (ointment and gel), the rats were euthanized by injecting a lethal dose (200 mg/kg) of pentobarbital, and the abdominal skin was removed. The FEL formulations were wiped off from the skin surface, and homogenized in methanol to extract FEL; the homogenates were centrifuged at 20,400 × g for 20 min at 4 °C. FEL concentrations in the supernatants were measured using HPLC as described above.

Statistical analysis

The sample numbers (n) are shown in figure legends, and data are expressed as the mean ± standard error (S.E.) of the mean. Statistical analysis was performed using the JMP ver. 5.1 (SAS Institute). Student’s t-test and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison were used for analyses. Statistical significance was set at P < 0.05.