Male ddy mice (5 weeks old; Japan SLC, Inc., Shizuoka, Japan) were maintained in the experimental animal facility of Meiji Pharmaceutical University. All mice were housed under standard conditions (23 ± 2 °C) with a 12:12-h light/dark cycle (lights off at 19:00). Food and water were provided ad libitum. After completion of relevant experiments, mice were euthanized by drawing blood and exsanguination from the descending aorta under isoflurane inhalation anesthesia. All procedures were approved by the Animal Care and Use Committee at Meiji Pharmaceutical University and conducted strictly in accordance with the National Institutes of Health guidelines.
Giotrif® tablets (afatinib maleate) were obtained from Boehringer Ingelheim Japan (Tokyo, Japan). Standard material for afatinib was obtained from SYNkinase (Melbourne, Australia). Minocycline hydrochloride was obtained from Sigma Aldrich (St. Louis, MO). White petrolatum (WP) was obtained from KENEI Pharm. Co., Ltd. (Osaka, Japan). Ammonium acetate was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Liquid chromatography-mass spectrometry (LC/MS) grade acetonitrile and deionized water were obtained from Wako Chemical Industry (Tokyo, Japan). All other chemicals were of analytical grade.
2.3. Evidence of afatinib-induced dermatitis in a mouse model
Mice were divided into five groups: group 1, control (n=4); group 2 as low concentration, 0.075% afatinib application (n=4); group 3, minocycline treatment after onset of the low conc. afatinib-induced rash (n=4); group 4 as high conc., 0.10% afatinib application (n=4); and group 5, minocycline treatment after onset of the high conc. afatinib-induced rash (n=4). The number of animals used in all experiments was as small as possible to produce valid results.
2.3.1 Preparation of the skin disorder mouse model by afatinib application
A skin disorder was induced by application of 0.075% or 0.10% (0.75 or 1.0 mg/g) afatinib petrolatum ointment (0.1 g/body respectively) as group 1,2 and 4. Observation was continued for 7 days under afatinib application.
2.3.2 Treatment of the skin disorder by simultaneous minocycline application
To estimate the concentration ratio between afatinib and 0.03% minocycline ointment (0.1 g/body), minocycline application started simultaneously with afatinib application as group 3 and 5.
2.4. Effects of minocycline ointment on oral afatinib administrations
Mice were divided into four groups: group 1, as control that mice received afatinib administration (20 mg/kg BW) at once a day for 18 days with vehicle (WP) pretreatment started 3 days before afatinib administration (n=4); group 2, afatinib administration (20 mg/kg BW) with minocycline WP pretreatment (n=4); group 3, as control that mice received afatinib administration (20 mg/kg BW) at once a day for 18 days with control treatment with vehicle (WP) after the afatinib-induced rash onset (n=4); and group 4, 0.03% minocycline WP treatment after the afatinib-induced rash onset (n=4). The number of animals used in all experiments was as small as possible to produce valid results.
Based on a result of the oral dose determination experiment, mice received around 0.6 mg/0.6 mL/30 g BW afatinib in 0.1% Tween 80 (20 mg/kg BW). For pretreatment of afatinib-induced skin disorders, application of vehicle (WP) or 0.03% (0.3 mg/g) minocycline WP ointment (0.1 g/body) was started 3 days before afatinib administration as group 1 and 2. On the other hand, vehicle (WP) or 0.03% minocycline application for the afatinib-induced rash started after the onset of the rash, about 10 days after oral administration as group 3 and 4.
2.5. Measurement of transepidermal water loss (TEWL)
TEWL was measured under isoflurane anesthesia on the dorsal skin lesion three times per week using a Tewameter® T210 (Courage + Khazaka Electronic GmbH, Cologne, Germany), and the median value of three measurements at each time point was recorded.
2.6. Tissue preparation and treatment of euthanasia
All mice were anesthetized with isoflurane (4% induction, 2% maintenance). Under inhalation anesthesia with isoflurane whole blood (ca. 1 mL) was collected from the descending aorta and then exsanguinated. Their dorsal skin lesions were quickly removed, washed with saline and stored frozen. The wet volumes of their dorsal skin samples were weighed. For histological analysis using light microscopy, a section of the dorsal skin was immersed in fixative containing 10% paraformaldehyde. After fixation, the dorsal skin was trimmed, washed with PB, dehydrated through graded concentrations of ethanol, cleared in xylene, and embedded in paraffin. Trough the above process, it is considered that mice could be euthanized by collecting whole body blood under the same effect as exsanguination under isoflurane inhalation anesthesia.
2.7. Histological analysis
For hematoxylin-eosin (HE) staining and to further investigate skin damage status, the dorsal skin blocks were cut into 5-µm-thick sections on a microtome (REM-710; Yamato, Japan). The sections were mounted on glass slides, deparaffinized with xylene, and immersed in degraded concentrations of ethanol. After washing in distilled water, several sections from each group were stained with HE solution. After washing, the sections were dehydrated through graded concentrations of ethanol, cleared with xylene, and cover slipped. The EGFR of keratinocytes in the dorsal skin samples from each group was incubated with anti-EGFR rabbit antibody (HPA018530, Atras Antibodies AB, Bromma, Sweden). After several washings, the sections were incubated with HRP-conjugated anti-rabbit IgG antibody produced in goat (A6454, Sigma, St. Louis, MO). After the DAB reaction procedures, the sections were cover slipped. All images of stained sections were captured using a CCD camera system (BZ-X700; Keyence, Japan) .
2.8. Chromatography conditions
Samples were analyzed using a Shimadzu LC/MS/MS system (LCMS-8040) with an electrospray source, coupled to a Shimadzu LC system (Tokyo, Japan). Separation was performed in an Inertsil ODS-3 (C18, 5 µm) 2.1 mm i.d. ´ 150 mm with a guard cartridge, 2.1 mm i.d. ´ 10 mm (GL Science Inc., Tokyo, Japan), maintained at 40 °C and eluted at a mobile phase flow rate of 0.2 mL/min. The isocratic mobile phase consisted of 5 mM ammonium acetate and acetonitrile (50:50 v/v). The MS conditions were: electrospray ionization, positive mode; capillary voltage, 4.5 kV; nebulizer gas, nitrogen at a flow rate of 1.5 L/min; drying gas, nitrogen at a flow rate of 15 L/min; vaporizer temperature, 400 °C; and ion transfer capillary temperature, 300 °C. The scan time was set at 0.3 s per transition. The selected reaction monitoring transitions were m/z 486.1 to 371.1 for afatinib and m/z 446.9 to 128.1 for the internal standard gefitinib, and these were determined by scan mode and reference  . Standard curves were linear (r2 > 0.99) over the range of 1–600 ng/mL. The lower limit of quantification (LLOQ) of the method was 1 ng/mL. The extraction recovery for afatinib in plasma at 50 ng/mL was 80.62%. For detection of afatinib in plasma samples, the extraction recovery of afatinib at 1, 3, 300, and 480 ng/mL was found to be in the range of 74.47–84.52%. The intra- and inter-batch precisions (RSD %) and the intra- and inter-batch accuracies were within 15%.
2.9. Sample preparation
Plasma samples were separated from blood treated with anticoagulant by centrifugation at 3000 ´ g for 10 min. The obtained plasma was then deproteinized using an equal volume of acetonitrile and centrifuged at 15,000 ´ g for 15 min. An equal volume of gefitinib (0.1 µM) acetonitrile solution as internal standard was subsequently added to the supernatant and centrifuged at 15,000 ´ g for 15 min. Plasma samples were stable for at least 6 months at -30 °C.
2.10. Experimental outcomes
The primary experimental outcome is to improve skin damage, eliminate scabs and reduce TEWL levels as a result of minocycline ointment. The secondary outcome is microscopic improvement of skin tissue by HE staining.
2.11. Statistical analysis
Pharmacokinetic parameters were evaluated using analysis of variance (ANOVA). Probability values less than 0.05 were considered significant. Statistical analysis was performed with SPSS statistical software (SPSS Inc. Chicago, IL, USA). All parameter values are expressed as means ± SD.