Animals

Seven-week-old male Wistar rats and adult male Japanese White rabbits (weight: 2–3 kg) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). The study was also conducted following the ARRIVE guidelines. The animals were housed under standard conditions (7:00 a.m. to 7:00 p.m. under fluorescent light, 7:00 p.m. to 7:00 a.m. in the dark, at 25 °C). Water, CE-2 (rat), and CE-3 (rabbit) standard diets (Clare Japan, Tokyo, Japan) were freely provided. Animal experiments were conducted following the Pharmacy Committee Guidelines for the Care and Use of Laboratory Animals in the Japanese Pharmacological Society and Kindai University (project identification code No. KASP-2021-003 and KASP-2021-004, approved on April 1, 2021).

Chemicals

BRI powder was purchased from Nacalai Tesque (Kyoto, Japan). Mannitol (D-mannitol), tyloxapol, methyl p-hydroxybenzoate, and isoflurane were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Methylcellulose (MC, type SM-4) with an average viscosity of approximately 4 mPa·s at 20 °C was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Benzalkonium chloride (BAC) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and 2-Hydroxypropyl-β-cyclodextrin (HPβCD) was from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Azopt® (CA-BRI) was obtained from Novartis Pharma K.K. (Tokyo, Japan) and Benoxil® ophthalmic solution (oxybuprocaine hydrochloride) was from Santen Pharmaceutical Co., Ltd. (Osaka, Japan). All other chemicals used were commercially available in special grade or HPLC grade.

Preparation of BRI nanocrystal formulations

BRI microcrystal formulation (BRI-MPs) was prepared by mixing 1% BRI powder, 0.8% MC (dispersant), 0.5% D-mannitol (stabilizer), and 0.001% BAC (preservative) in 5.0% HPβCD (solubilizer) aqueous solution, and BRI nanocrystal formulation (BRI-NPs) was prepared by bead-milling the pre-prepared BRI-MPs. The bead-milling was performed under the following conditions using zirconia beads (diameter: 0.1 mm) and Bead Mill NP-100 (Thinky Co., Ltd., Tokyo, Japan): 2,000 rpm for 180 s × 8 times at -20 °C, followed by 400 rpm for 60 s at -20 °C. After milling, we collected the desired suspension, taking care not to take precipitated zirconia beads. The BRI nanocrystal formulation with tyloxapol (Tyl@BRI-NPs) was prepared by adding 0.0005% tyloxapol to BRI-NPs and the BRI microcrystal formulation with tyloxapol (Tyl@BRI-MPs) was also provided using the same protocol.

Particle size and morphology

The particle size of BRI ophthalmic formulations was measured with the laser diffraction particle size analyzer SALD-7100 (Shimadzu Corp., Kyoto, Japan) and NANOSIGHT LM10 (Malvern, Worcestershire, UK) utilizing nano tracking analysis. The refractive index was set at 1.95 ± 0.05i in SALD-7100, and the conditions for NANOSIGHT LM10 were as follows: wavelength, 405 nm; measurement time, 60 s; viscosity, 1.27 mPa∙s. The particle morphology was observed using the scanning probe microscope SPM-9700 (Shimadzu Corp., Kyoto, Japan) and a scanning electron microscope (SEM, NeoScope JCM-7000, JEOL Ltd., Tokyo, Japan).

Powder X-ray diffractometry (PXRD)

PXRD was performed using MiniFlex 600 (Rigaku Corp., Tokyo, Japan) as the X-ray generator for Cu Kα radiation was used. The samples were prepared by filtering the suspension and drying the filter residue under reduced pressure at room temperature. The PXRD data were collected in the continuous one-dimensional scanning mode with a step size of 0.02 degrees. The scanning speed was 3 degrees/min and the scanned range was 5 to 50 degrees.

Thermogravimetry and Differential thermal analysis (TG-DTA)

TG-DTA was performed using DTG-60 H (Shimadzu Corp., Kyoto, Japan), under a nitrogen-rich atmosphere (flow rate: 50 mL/min) in aluminum pans. The samples were prepared by filtering the suspension and drying the filter residue under reduced pressure at room temperature. Prepared 5 mg samples were heated from room temperature to 200 °C at a heating rate of 10 °C/min.

Determination of BRI concentration by HPLC

The concentrations of BRI were determined using an LC-10AD system (HPLC, Shimadzu Corp., Kyoto, Japan) using 0.1 µg/mL methyl p-hydroxybenzoate as an internal standard. Inertsil ODS-3 (2.1 mm × 50 mm, GL Science Co., Inc., Tokyo, Japan) was used and the column temperature was 35 °C. The mobile phase consisted of 0.1 M potassium phosphate and acetonitrile (85/15) pumped at a flow rate of 0.25 mL/min for 16.5 min. The detection wavelength was 250 nm. When measuring under the above conditions, the retention time for internal standard and BRI are 3–4 min and 7.5–8.5 min, respectively. The limit of detection (LOD) and quantification (LOQ) of BRI is 0.004 µg/mL and 0.5 µg/mL, respectively.

Viscosity

Viscosity was measured using a tuning fork vibration viscometer, SV-1 A (A&D Co., Ltd., Tokyo, Japan) at 26 °C. Each formulation was put into a 2 mL tube and the oscillator drive frequency was set to 30 Hz for the measurements.

Zeta potential

The zeta potential was measured using a micro-electrophoresis zeta potential analyzer (Sanyo Trading Co., Ltd., Tokyo, Japan). Diluted each formulation 20 times with water and measured under the following conditions: electrophoresis distance, 120 μm; strength of electric field, 10 V/cm; sample temperature, 26 °C.

Dispersal stability for BRI ophthalmic formulations

The experiment was performed according to our previous report [7]. Two milliliters of samples were placed in 3 mL test tubes and incubated in the dark at 20 °C for 28 days. At each sampling point (days 0, 1, 3, 7, 14, and 28), digital images were taken and, after 28 days, the particle size was measured by NANOSIGHT LM10.

In vitro transcorneal penetration study by using rat cornea

The rats were sacrificed by injecting a lethal dose of pentobarbital (200 mg/kg), and their corneas were removed and placed on a Franz diffusion cell (12.2 mL, Osaka-riko Co., Ltd., Osaka, Japan). The reservoir chamber of the cell was filled with 10 mM HEPES buffer (pH 7.4) containing 136.2 mM NaCl, 5.3 mM KCl, 1.0 mM K2HPO4, 1.7 mM CaCl2, and 5.5 mM glucose, and 20 µL of each BRI ophthalmic formulation was applied into the top of the cell. The experiments were performed at 37 °C. At predetermined times (0–60 min), 200 µL of the HEPES buffer at the reservoir chamber was sampled and immediately replenished with the same volume of buffer in the reservoir chamber. The concentration of BRI in the collected samples was measured under the HPLC conditions described above, and the area under the drug concentration-time curve (AUC0-60 min) was calculated.

In vivo transcorneal penetration study

The in vivo transcorneal penetration study of the 1% BRI ophthalmic formulations was determined following our previous reports [7]. Rabbits were anesthetized with isoflurane (flow rate 1.0 L/min, set concentration 3%), and an injection needle (26 G) with a silicon tube (diameter: 0.5 mm) was inserted just below the cornea. After 50 µL of BRI ophthalmic formulations was applied, 5 µL of aqueous humor was collected at predetermined times (0–90 min) and the concentration of BRI in the aqueous humor was measured under the HPLC conditions described above to determine the area under the drug concentration-time curve in the aqueous humor (AUC0-90 min) according to the trapezoidal rule up to the last BRI concentration measurement point.

Assessment of corneal damage

Fifty microliters of BRI ophthalmic formulations were instilled in rabbits twice a day (10:00 and 18:00) for one week. Afterward, the rabbit corneas were stained with a solution containing 1% fluorescein and 0.4% oxybuprocaine hydrochloride, and the corneal surface was observed by using a slit lamp METORI-50 V (Seed Corporation, Saitama, Japan) with a blue filter.

Evaluation of IOP

Ocular hypertension in rabbits was induced as a rapid intravenous injection of 15 mL/kg glucose solution. After 50 µL of 1% BRI ophthalmic formulations were applied, the IOP of rabbits was measured at predetermined times (0–90 min) using an electronic tonometer (Medtronic SOLAN, Jacksonville, FL, USA). IOP was measured thrice repeatedly at a one-time point, and the average IOP at each time point was calculated. ΔIOP was calculated from the difference between normal IOP and IOP after the administration of BRI ophthalmic formulations, and the area under the curve of ΔIOP versus time (AUCΔIOP) was calculated according to the trapezoidal rule up to the last IOP measurement point.

Statistical analysis

The particle size data from SALD-7100 are presented as the mean ± standard deviation, and other data are presented as the mean ± standard error. In this study, Student’s t-test and Dunnett’s multiple comparison were used to determine significant differences, and a P value less than 0.05 was considered significant.