Preparation of an Ultrafine Rebamipide Ophthalmic Suspension with High Transparency

Abstract

A 2% commercially available, milky-white, rebamipide micro-particle suspension is used to treat dry eyes, and it causes short-term blurring of the patient’s vision. In the current study, to improve the transparency of a rebamipide suspension, we attempted to obtain a clear rebamipide suspension by transforming the rebamipide particles to an ultrafine state. In the initial few efforts, various rebamipide suspensions were prepared using a neutralizing crystallization method with additives, but the suspensions retained their opaque quality. However, as a consequence of several critical improvements in the neutralizing crystallization methods such as selection of additives for crystallization, process parameters during crystallization, the dispersion method, and dialysis, we obtained an ultrafine rebamipide suspension (2%) that was highly transparent (transmittance at 640 nm: 59%). The particle size and transparency demonstrated the fewest level of changes at 25°C after 3 years, compared to initial levels. During that period, no obvious particle sedimentation was observed. The administration of this ultrafine rebamipide suspension (2%) increased the conjunctival mucin, which was comparable to the commercially available micro-particle suspension (2%). The corneal and conjunctival concentration of rebamipide following ocular administration of the ultrafine suspension was slightly higher than that of the micro-particle suspension. The ultrafine rebamipide suspension (eye-drop formulation) with a highly transparent ophthalmic clearness should improve a patient’s QOL by preventing even a shortened period of blurred vision.

Rebamipide, 2-(4-chlorobenzoylamino)-3-(2-oxo-1,2-dyhydroquinolin-4-yl) propanoic acid, was developed by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan), and has been used clinically in Asian countries including Japan, as well as in Egypt, to promote the healing of gastric ulcers as well as to prevent ulcer recurrence.^1,2) In the ophthalmologic field, a 2% rebamipide ophthalmic suspension (Mucosta^® ophthalmic suspension UD2%) is known to be effective for the treatment of dry eyes,^3,4) and, therefore, has been marketed in Japan for the treatment of this condition since January 2012. Rebamipide increases the mucin-like glycoproteins^5,6) and suppresses cytokine production in the cornea and conjunctiva,^7,8) which results in improvements in symptoms related to dry eyes.

The solubility of rebamipide is dependent on its pH due to the carboxylic groups in the molecular structure (Fig. 1). However, the commercially available rebamipide micro-particle suspension (2%) was formulated under pH 5.5–6.5 as a white milky aqueous suspension⁹⁾ containing fine rebamipide crystals (mean particle size: approximately 1 µm), because an almost neutral pH solution is suitable for the treatment of patients with dry eyes. This suspension must be shaken well for redispersion prior to placing a drop in the eye since the suspension forms a precipitate layer during storage. A patient who is administered such a milky white suspension will suffer from a blurring of vision shortly after a drop is placed in the eye. In addition, this suspension cannot be sterilized by sterile filtration with a 0.2 µm filter, which causes problems from a manufacturing point of view. These disadvantages of the commercially available micro-particle suspension must be overcome. However, no obvious method is available to produce a high transparency rebamipide suspension suitable for ophthalmic use.

Fig. 1. Chemical Structure of Rebamipide

Ultrafine drug crystals are generally manufactured using either a bottom-up or a top-down approach.¹⁰⁾ Wet media milling is widely used in the top-down version.^11–16) Most nano-suspensions prepared by media bead milling, however, remain opaque and impossible to sterilize via filtration, because in general this method cannot reduce the crystal size to less than 100–200 nm.¹³⁾ Another disadvantage of wet media milling is the contamination from collisions among medias.¹⁰⁾ Therefore, in the present study, we focused on the bottom-up procedure to obtain an ultrafine rebamipide suspension that would be highly transparent. Some of the nanoprecipitation techniques that use the bottom-up procedure include solvent precipitation,¹⁷⁾ neutralizing crystallization,^18,19) supercritical fluid technologies,²⁰⁾ and controlled evaporation of droplets.²¹⁾ Hirakawa et al. reported the use of neutralizing crystallization to prepare a fine-grained oxolinic acid. Their method comprised dissolving oxolinic acid with a base, and neutralizing it by an acid in the presence of a polymer compound and/or a surfactant.¹⁸⁾ With this procedure, they prepared phenytoin and phenobarbital formulations with an average particle size of 3 to 4 µm.¹⁹⁾ The size of the crystals could depend on the physicochemical properties of a compound, which could generally make it difficult to control the size of the crystals via a neutralizing of the crystallization. Nevertheless, in the current study, we chose to use an improved method to neutralize the crystallization to prepare an ultrafine rebamipide suspension with an enhanced transparency. Furthermore, in order to evaluate the effect of the ultrafine rebamipide suspension on dry-eye-related symptoms, the corneal and conjunctival concentrations of rebamipide following ocular administration was determined and the results were compared with the commercially available micro-particle suspension. We also tested the amount of mucin in the conjunctiva of rabbits following ocular administration.

MATERIALS AND METHODS

Materials

A bulk rebamipide powder manufactured by Otsuka Pharmaceutical Co., Ltd., was used as the active ingredient. We used a 2% rebamipide micro-particle suspension manufactured by Otsuka Pharmaceutical Co., Ltd., because it was the same formulation as a Mucosta^® ophthalmic suspension UD2%. Hydroxypropylmethylcellulose (HPMC) (TC-5 grade or 60SH50 grade, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan), polyvinylpyrrolidone K25 or K90 (PVP K25 or PVP K90) (BASF AG, Land Rheinland-Pfalz, Germany), polyoxyethylene [160] polyoxypropylene [30] glycol (Pluronic^® F68, BASF), carboxymethylcellulose (CMCNa) (Serogen F7-A grade, DKS Co., Ltd., Kyoto, Japan), polysorbate 80 (TO-10M, Niko Chemical, Tokyo, Japan), and macrogol 4000 (NOF Corporation, Tokyo, Japan) were used.

Animals

Healthy New Zealand White (Kitayama Labes Co., Ltd., Ina, Japan) male rabbit/Kbs were used. The rabbits were housed at a temperature of 23±2°C under a humidity of 55±25%, with a 12-h light-dark cycle, and were allowed access to food (LRC4, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. All experiments were carried out in accordance with the “Otsuka Pharmaceutical Guidelines on Animal Experiments,” which complied with the “Principles of Laboratory Animal Care” (NIH publication No. 85–23, revised in 1985).

Preparation of Ultrafine Rebamipide Suspensions

A 1-L scale of 2% ultrafine rebamipide suspensions was prepared, as described below and illustrated in Fig. 2.

An acidic additive solution and a basic rebamipide solution were prepared separately (Step-1 in Fig. 2). For the acidic additive solution, a predetermined amount of the additive (polymers or surfactants listed in Table 1) and 12 mL of concentrated hydrochloric acid (14.2 g) were dissolved in purified water to obtain 280 mL of solution. For the basic rebamipide solution, 4.4 g of sodium hydroxide and 20 g of rebamipide were dissolved in purified water with heating at 40°C to obtain 700 mL of solution.

Fig. 2. Flow Diagram of the Preparation Process for a 1L Scale of an Ultrafine Rebamipide Suspension

* In the case of 1% additive. ** For the eye-drop formulation, the crystallization was carried out in a rotary homogenizer (rate of rotation: 10000 rpm). *** Dispersion mechanism of the high-speed liquid-liquid shearing method (CLEARMIX^® W-MOTION).

Table 1. Primary and Secondary Particle Sizes and Transmittance in the Crystalized Suspension (Intermediate-1 in Fig. 2) and Dispersed Crystalized Suspension Using a Rotary Homogenizer at 10000 rpm for 10 min

Additive*	Crystalized suspension			Dispersed crystalized suspension
	Primary particle size** (µm)	Secondary particle size† (µm)	Transmittance‡ (%)	Primary particle size** (µm)	Secondary particle size† (µm)	Transmittance‡ (%)
None	1.0	6.7	0.1	0.34	0.62	0.1
HPMC (TC-5)	8.7	20.3	5.1	0.20	29.0	63.9
PVP K25	3.8	45.8	25.5	0.21	0.86	34.4
Pluronic F68	0.17	0.39	23.8	0.19	0.37	24.3
CMCNa	0.15	0.35	4.3	0.16	0.16	5.7
Macrogol 4000	0.20	7.5	0.5	0.24	7.0	0.4
Polysorbate 80	0.34	4.2	2.3	0.34	6.3	1.3
HPMC (60SH50)	12.1	20.2	0.5	7.6	9.2	0.5
PVP K90	12.7	20.1	0.3	11.2	11.7	0.4

n=1. * One % (w/v) of additive was added in the crystalized suspensions except for HPMC (60-SH50), which were added at 0.5%. ^** Primary particle size was measured in a circulatory cell under ultrasonic wave. 0.2% (w/v) HPC-SL solution was used as a dispersion medium. ^† Secondary particle size was measured in a batch cell without ultrasonic wave. Purified water was used as a dispersion medium. ^‡ Transmittance in the 10-fold diluting suspensions was measured at 640 nm with a spectrophotometer.

The rebamipide was precipitated by slowly adding the basic rebamipide solution into the acidic additive solution (Step-2 in Fig. 2). The precipitated suspension was let stand overnight, and then the pH of the mixture was further adjusted to 6 using a 5 M sodium hydroxide solution. The 2% rebamipide crystalized suspension (Intermediate-1 in Fig. 2) was obtained via dilution with purified water.

A 2% rebamipide crystalized suspension, prepared with the additive, was further dispersed using a CLEARMIX^® W-MOTION (M-Technique Co., Ltd., Osaka, Japan) with a rotor and screen that turned at approximately 18000 and 16000 rpm, respectively (Step-3 in Fig. 2). The 2% rebamipide dispersed suspension (Intermediate-2 in Fig. 2) was concentrated and desalted via dialysis (Pellicon^® XL50, Merck Millipore, Massachusetts, U.S.A.) (Step-4 in Fig. 2). Following dialysis, a 2% ultrafine rebamipide suspension was finally obtained via dilution with purified water.

In the preparation of an eye-drop formulation, the precipitation of rebamipide (Step-2 in Fig. 2) was conducted using an inline type rotary homogenizer (CLEARMIX^® Single-MOTION, M-Technique) with a rotor that turned at 10000 rpm to further improve the transparency of the rebamipide suspension. The isotonic pressure of the rebamipide suspension was adjusted via the addition of glycerin. This suspension was filtered using a sterile filter with a pore size of 0.2 µm (EKV filter, Pall Corporation, NY, U.S.A.) to obtain an eye-drop formulation.

Evaluation of Rebamipide Suspensions

A. Particle Size

The particle size of the crystalized suspension was measured using a Laser Diffraction Particle Size Distribution Analyzer (SALD 3000J, Shimadzu Corporation, Kyoto, Japan). To establish the particle distribution, the volume mean diameter was measured with ultrasonic irradiation in a circulating cell using a 0.2% HPC-SL solution as the dispersion medium, which recorded as the primary particle size. The volume mean diameter was measured without ultrasonic irradiation in a batch cell using purified water as the dispersion medium, and was recorded as the secondary particle size. The average particle size (z-average size) of the dispersed suspension along with the ultrafine suspension was measured using a Dynamic Light Scattering Particle Size Analyzer (Nano-ZS, Malvern, Worcestershire, U.K.).

B. Crystal Shapes

The 2% rebamipide ultrafine crystalized suspensions prepared with either 1% HPMC (TC-5) or 1% PVP K25 were mixed with an equal amount of 4% uranyl acetate solution and then dried. The crystal shape of the sample was observed via transmission electron microscope (JEM-1200EX, JEOL Ltd., Tokyo, Japan).

C. X-Ray Diffraction Spectra

The 2% rebamipide ultrafine crystalized suspensions prepared using either 1% HPMC (TC-5) or 1% PVP K25 were ultra-centrifuged using a Beckman L7-Ultracentrifuge (50000 rpm, for 60 min, 10°C) to precipitate a fine grain. Then, the precipitate was washed with purified water and ultra-centrifuged again. The resultant precipitate was then air-dried. X-Ray diffraction spectra of the dried precipitate were measured via X-ray diffraction measurement (D8 ADVANCE, BRUKER Massachusetts, U.S.A.).

D. Transparency

All of the 2% crystalized suspensions remained opaque. Hence, the 2% crystalized suspension was diluted 10-fold with purified water. The transmittance of the sample was measured at 640 nm using a spectrophotometer (UV-240, Shimadzu). On the other hand, the transmittance of the 2% ultrafine rebamipide suspension was measured without dilution.

Effects of Rebamipide Suspensions on the Amount of Mucin-Like Substances on the Conjunctiva of a Normal Rabbit

Either a 2% rebamipide micro-particle suspension or an ultrafine rebamipide suspension (0, 0.1, 0.3, 1 or 2%) was administered in both eyes of 11–12 week-old male rabbits 6 times a day for 14 d and once on day 15. After the final administration, the conjunctiva was excised and conjunctival mucin-like substances were measured using an Alcian blue binding method.⁶⁾ The rabbits were euthanized by intravenous injection of an excessive amount of pentobarbital sodium solution (50 mg/mL). Whole conjunctivae were removed from each rabbit, which were then weighed and maintained in a cold 0.25 M sucrose solution (10 mL). Then, the conjunctivae were incubated in 10 mL of a 0.1% Alcian blue solution that was dissolved in a buffer (0.15 M sucrose and 0.05 M sodium acetate, pH 5.8) at room temperature for 1.5 h. The conjunctivae were then washed twice with a 0.25 M sucrose solution (10 mL) and incubated in 10 mL of a 0.5 M MgCl₂ solution at room temperature for 2 h to extract the dye bound to the mucin layer of the conjunctiva. The obtained extract was mixed with 10 mL of diethyl ether. The optical density of the aqueous layer of the extract was measured at 605 nm. The amount of bound Alcian blue dye represented the optical density per unit weight of conjunctival tissue (O.D. units/g of conjunctival tissue). The amount of bound Alcian blue dye shown in the results refers to the mean value of both eyes. All values represent the mean±standard error (S.E.). Linear regression analysis confirmed that the mucin-like substances on the conjunctiva in the rebamipide ultrafine suspension group showed no monotonous increase with the logarithmic concentration of rebamipide; this analysis was followed by a two-tailed Dunnett test. A p value of less than 0.05 was considered statistically significant. Statistical analysis was performed using SAS software (Release 9.1, SAS Institute Japan). The 2% rebamipide micro-particle suspension group and the 2% ultrafine rebamipide suspension (eye-drop formulation) group were analyzed statistically in pairs in order to examine the equivalence of these groups for the amount of mucin-like substances on the conjunctiva. A one-way ANOVA was conducted using the values of the amount of mucin-like substances on the conjunctiva in each group to calculate common variance. Subsequently, the 90% confidence intervals for the difference of the average of the amount of mucin-like substances on the conjunctiva between the two groups were calculated. The tolerance intervals for the equivalence of the amount of mucin-like substances on the conjunctiva were set at ±20% of the mean of the amount of mucin-like substances on the conjunctiva in the 2% rebamipide micro-particle suspension group.

Concentration of Rebamipide in the Corneal and Conjunctivae

A single dose of rebamipide (2% ultrafine rebamipide suspension or 2% rebamipide micro-particle suspension) was administered to both eyes of 11–12 week-old male rabbits at a volume of 50 µL/eye. At 0.25, 1, 2, 4, 8, and 24 h after administration, the rabbits were euthanized via an overdose of pentobarbital sodium solution (50 mg/mL) in the auricular vein. Both eyeballs, as well as the eyelids, were harvested from individual 4 animals (7–8 eyes) at each time point and washed once in a container containing cold 0.9% physiological saline (Otsuka Pharmaceutical Factory Inc., Naruto, Japan). Each cornea and conjunctiva (bulbar conjunctiva and palpebral conjunctiva) was isolated and collected and then each sample was washed twice with cold 0.9% physiological saline. Each of the samples of cornea and conjunctiva was frozen with liquid nitrogen and then stored in an ultra-deep freezer (−80°C) until evaluation. The samples of cryopreserved cornea and conjunctiva were cut finely with ophthalmic scissors and then put in polypropylene tubes. These samples were homogenized in the presence of 0.9% physiological saline with Polytron (PT-MR2100, Kinematica AG, Luzern, Switzerland). The rebamipide concentrations in the cornea and conjunctiva samples were measured via LC-electrospray ionization (ESI) -MS/MS at the Sekisui Medical Co., Ltd. (Ibaraki, Japan).²²⁾

RESULTS

Crystallization of Rebamipide with Several Additives

The 2% crystalized rebamipide suspensions (Intermediate-1 in Fig. 2) were prepared using the neutralizing crystallization method (Step-1 and Step-2 in Fig. 2) with and without additives [low or middle molecular weight HPMC (TC-5 or 60SH50), PVP K25, PVP K90, Pluronic^® F68, CMCNa, macrogol 4000, polysorbate 80]. The Pluronic^® F68, CMCNa, macrogol 4000, and polysorbate 80 all produced less than 0.4 µm of a 2% crystalized rebamipide suspension (Intermediate-1) (Table 1) of primary mean particles. These particles, however, still appeared as a white milky suspension (data not shown). The primary particles were smaller in size than the secondary particles, which indicated that aggregated particles remained in the crystalized suspension. Low or middle molecular weight HPMC (TC-5 or 60SH50), PVP K25, PVP K90 resulted in larger primary particle sizes than no additive (1.0 µm). However, low molecular weight grade HPMC (TC-5) and PVP (K-25) showed higher transmittance than no additive.

Dispersion with a High-Speed Rotary Homogenizer

In order to disperse the aggregated particles, the crystalized suspensions (Intermediate-1) were further dispersed by a rotary homogenizer at 10000 rpm for 10 min. The mechanical dispersion decreased the primary particle size of the crystalized suspensions containing HPMC (TC-5) and PVP K25 to 0.2 µm and improved their transmittance (Table 1). On the other hand, the enhanced dispersion changed neither the primary particle size nor the transmittance of the crystalized suspensions containing Pluronic^® F68, CMCNa, macrogol 4000, polysorbate 80, middle molecular weight HPMC (60SH50) or high molecular weight PVP (K90). These results indicated the type and molecular weight of the additives affects the particle size, particle aggregation, and transparency. Accordingly, the crystalized suspensions containing TC-5 and PVP K25 were adopted for further studies.

Strong Dispersion Using a High-Speed Liquid–Liquid Shearing Method on the Crystalized Rebamipide Suspension Containing TC-5 and PVP K25

In order to further disperse the crystalized particles, a high-speed liquid–liquid shearing method was applied (Step-3 in Fig. 2, Intermediate-2). This forceful dispersion method decreased the z-average size of the crystalized rebamipide suspension containing TC-5 from 170–210 to 100–110 nm as well as that of a suspension containing PVP K25 from 100 to 90 nm (Fig. 3A). In addition, this dispersion method increased the transmittance of the crystalized rebamipide suspension containing 1–3% TC-5. Also, the suspension became semi-translucent (Fig. 3B). However, the transmittance of the crystalized rebamipide suspension containing 1–3% PVP K25 was not improved by this forceful dispersion method.

Fig. 3. Change in the Z-Average Size (A) and Transmittance (B) in 2% Dispersed Rebamipide Suspensions (Intermediate-2) via High-Speed Liquid–Liquid Shearing

(A) Change in the z-average size, (B) Change in the transmittance, The 2% crystalized rebamipide suspension (Intermediate-1), prepared with 1–3% TC-5 or 1–3% PVP K25, was dispersed using a CLEARMIX^® W-MOTION with the rotor and screen that turned at approximately 18000 and 16000 rpm, respectively. n=1.

Inhibiting Effect of Dialysis on the Aggregation of an Ultrafine Rebamipide Suspension

A prepared 2% dispersed rebamipide suspension containing 1% HPMC (TC-5) (Intermediate-2 after Step-3) formed a highly viscous gel during storage at 60°C for 2 weeks. This could have been due to the presence of the NaCl as a by-product during neutralization (Step-2). To eliminate the NaCl, dialysis was performed (Step-4). The dialysis prevented gelation of the prepared 2% ultrafine rebamipide suspension during storage at 60°C (Table 2). In addition, dialysis (Step-4) increased the transmittance of the prepared 2% dispersed rebamipide suspension containing 1% TC-5 from 16 to 45% (Table 3). Consequently, throughout Steps 1–4, the 2% ultrafine rebamipide suspension containing 1% TC-5 was prepared with better transmittance.

Table 2. Change in the Z-Average Size and Appearance of a 2% Ultrafine Rebamipide Suspension Containing 1% HPMC (TC-5) Stored at 60°C

Storage Condition	Dialysis	Z-Average size(nm) *		Appearance 2 weeks later
		1 week later	2 weeks later
60°C	None	402	459	Gelling
	Yes	186	213	Unchanged

n=1, * Z-average size at initial: before dialysis: 158 nm, after dialysis: 122 nm.

Crystal Morphology and Crystal Form of the Obtained Ultrafine Rebamipide Suspensions

The crystal shapes of the prepared 2% ultrafine rebamipide suspensions with either 1% HPMC (TC-5) or 1% PVP K25 were observed via transmission electron microscope. As shown in Fig. 4A, the crystal shape of the ultrafine rebamipide with 1% TC-5 resembled that of a homogenous hyper-needle crystal. A longer-gage length was from 300 to 1000 nm, and the shorter gage length was about 15 nm. On the other hand, the crystal shape of the ultrafine rebamipide with 1% PVP K25 was a homogenous needle crystal (Fig. 4B). The longer-gage length was approximately 200 nm, and the shorter-gage length was approximately 40 nm.

Fig. 4. Photographs of the Ultrafine Rebamipide Suspension via Transmission Electron Microscope

(A) Crystal shape of the ultrafine rebamipide with 1% TC-5, (B) Crystal shape of the ultrafine rebamipide with 1% PVP K25. One representative picture from 3 independent samples is shown. Bar indicates 200 nm.

The X-ray diffraction spectra of these ultrafine rebamipide crystals were also observed (Fig. 5). The positions of the major peaks in the X-ray diffraction spectra were the same as those of the bulk rebamipide powder. These results show that the two examples of ultrafine rebamipide crystals prepared in this study had the same crystal form as that of bulk rebamipide powder, which is commercially available for clinical use.

Fig. 5. X-Ray Diffraction Spectra of Ultrafine Rebamipide Crystals or Bulk Rebamipide Powder

(A) X-Ray diffraction spectra of the ultrafine rebamipide with 1% HPMC (TC-5), (B) X-Ray diffraction spectra of the ultrafine rebamipide with 1% PVP K25, (C) X-Ray diffraction spectra of bulk rebamipide powder. One representative data from 3 independent samples is shown.

Preparation of an Eye-Drop Formulation Using the Ultrafine Rebamipide Suspension

We prepared an eye-drop formulation (2% ultrafine rebamipide suspension with 1% HPMC (TC-5) and 2.35% glycerin) by further improvement to the neutralized crystallization (Step-2) via an inline type rotary homogenizer followed by dispersion and dialysis. Homogenization (10000 rpm) during crystallization (Step-2) further improved the transparency of the eye-drop formulation (transmittance became 59%, Table 3). In addition, the eye-drop formulation was capable of passing through a sterile filter with a pore size of 0.2 µm. Filtration did not lower the potency (Table 4). A 2% ultrafine rebamipide suspension (eye-drop formulation) in a unit-dose eye-drop container after sterilization is shown in Fig. 6. The eye-drop formulation was much more transparent than the commercially available micro-particle suspension.

Table 3. The Z-Average Particle Size and the Transmittance at 640 nm in a 2% Ultrafine Rebamipide Suspension Containing 1% HPMC (TC- 5) before and after Dialysis

	Z-Average size (nm)	Transmittance at 640 nm in 2% suspension
Dispersed suspension before dialysis	126	16.2
After dialysis	104	45.0
Eye-drop formulation*	106	59.0

n=1, * Eye-drop formulation was prepared via crystallization in an in-line type rotary homogenizer (rate of rotation: 10000 rpm) followed by dispersion and then dialysis.

Table 4. Change in the Potency of a 2% Ultrafine Rebamipide Suspension before or after Filtration through a Pore Size of 0.2 µm (EKV Filter)

	Before filtration	After filtration
Potency*	102.1%	101.1%

n=1, * Twenty liters of 2% ultrafine rebamipide suspension were filtered using a sterile filter with a pore size of 0.2 µm (10 inches of EKV filter, Pall). The concentrations of rebamipide in the ultrafine rebamipide suspension were measured before or after filtration, and then the potency was calculated by the following formula: Potency (%)=the concentration (%) of rebamipide/2×100.

Fig. 6. Appearance of the Commercially Available Rebamipide Micro-particle Suspension (Left Side) and the 2% Ultrafine Rebamipide Suspension (Eye-Drop Formulation) (Right Side), Which Was Filled in a Unit-Dose Eye-Drop Container

The change in the z-average size and transmittance of the eye-drop formulation during storage was evaluated (Fig. 7). At 40°C for 6 months, the z-average size was slightly increased and transmittance was slightly decreased. No change in appearance, such as the sedimentation of particles, was observed. At 25°C for 36 months, only small changes were observed in the z-average size, transmittance and appearance. These results indicate that the eye-drop formulation was stable for a long period of time without causing either aggregation or sedimentation of the particles at room temperature.

Fig. 7. Change in the Z-Average Size and Transmittance in the 2% Ultrafine Rebamipide Suspension (Eye-Drop Formulation) Stored at 25°C and 40°C

(A) Change in the Z-Average size with storage period, (B) Change in the transmittance with storage period. The Z-Average size was measured three times and values represent the mean±standard deviation (S.D.). The transmittance was measured single time.

Effects of an Ultrafine Rebamipide Suspension on the Amount of Mucin-Like Substances on the Conjunctiva of Rabbits

To evaluate the efficacy of the 2% ultrafine rebamipide suspension (eye-drop formulation) for the treatment of keratoconjunctivitis sicca, either the eye-drop formulation or a commercially available rebamipide micro-particle suspension was applied to the eyes of the rabbits, and the amount of mucin-like substances on the conjunctiva of the rabbits’ eyes was measured using the Alcian blue binding method. In the 2% ultrafine rebamipide suspension (eye-drop formulation) group, the amount of mucin-like substances on the conjunctiva was 0.276±0.007 O.D. units/g of conjunctival tissue. The treatment obviously increased the amount of mucin-like substances on the conjunctiva compared with that of the control group (Fig. 8). In the commercially available 2% rebamipide micro-particle suspension group, the amount of mucin-like substances on the conjunctiva was 0.261±0.007 O.D. units/g of conjunctival tissue. The conjunctival mucin-increasing action was equivalent between our eye-drop formulation and the commercially available 2% rebamipide micro-particle suspension, since the 90% confidence intervals for the difference of the average of the amount of mucin-like substances on the conjunctiva between the 2% rebamipide micro-particle suspension and our eye-drop formulation were −0.031 to 0.003 O.D. units/g of conjunctival tissue, and the ratios of these values to the mean value in the 2% rebamipide micro-particle suspension were from −11.9 to 1.0%. We further prepared several eye-drop formulations (0, 0.1, 0.3, or 1%) and applied these to the rabbits’ eyes. The eye-drop formulation increased the amount of mucin-like substances on the conjunctiva in a dose-dependent manner.

Fig. 8. Effects of the 2% Rebamipide Micro-Particle Suspension and the 0, 0.1, 0.3, 1 or 2% Ultrafine Rebamipide Suspension (Eye-Drop Formulation) on the Amounts of Mucin-Like Substances in the Conjunctiva of New Zealand White Male Rabbits

Each formulation was administered in both eyes of rabbits 6 times a day for 14 d and once on day 15 at doses of 50 µL/eye. Each bar represents the mean±S.E., n=8 (One value of the amount of bound Alcian Blue dye shown in the results refers to the mean value of both eyes.). ** p<0.01 vs. the 0% ultrafine rebamipide suspension (Dunnett test (two-tailed)).

Distribution of Corneal and Conjunctival Rebamipide in the Rabbits after Ocular Administration

The corneal and conjunctival concentrations of rebamipide following ocular administration of the 2% ultrafine rebamipide suspension (eye-drop formulation) or commercially available 2% rebamipide micro-particle suspension were measured in the rabbits. In the micro-particle suspension group, rebamipide concentration reached a maximum corneal level (0.33 µg/g tissue) at 1 h and a maximum conjunctival level (1.13 µg/g tissue) at 0.25 h, respectively (Fig. 9). The values for area under the concentration–time curve from time zero to t hours (AUC_t) in the cornea and conjunctiva were 2.61 and 14.1 µg·h/g, respectively (Table 5). In the eye-drop formulation, the concentration of rebamipide reached a corneal maximum (0.91 µg/g tissue) at 0.25 h and a conjunctival maximum (2.08 µg/g tissue) at 0.25 h. The values for AUC_t in the cornea and conjunctiva were 4.16 and 16.9 µg·h/g, respectively. The corneal and conjunctival concentrations of rebamipide following ocular administration of the eye-drop formulation was slightly higher than that of the micro-particle suspension, even though no significant difference was observed in the conjunctival mucin that would have increased the action between the 2% eye-drop formulation and the 2% rebamipide micro-particle suspension (Fig. 8).

Fig. 9. Corneal and Conjunctival Concentration Profiles of Rebamipide Following Single Ocular Administration of the 2% Rebamipide Micro-Particle Suspension and the 2% Ultrafine Rebamipide Suspension (Eye-Drop Formulation) in New Zealand White Male Rabbits at a Dose of 50 µL/Eye

(A) Corneal, (B) Conjunctival. After single administration into both eyes of 4 animals at a dose of 50 µL/eye, the corneal and conjunctival concentration of rebamipide were determined by LC/MS/MS at 0.25, 1, 2, 4, 8, and 24 h. Values represent the mean±S.D. (n=7–8 eyes from 4 animals).

Table 5. Pharmacokinetic Parameters of Rebamipide Following a Single Ocular Administration of a Commercially Available 2% Rebamipide Micro-particle Suspension and a 2% Ultrafine Rebamipide Suspension (Eye-Drop Formulation) to New Zealand White Male Rabbits at a Dose of 50 µL/eye

Parameter	Formulation	Cornea	Conjunctiva
Cmax (µg/g)	Rebamipide micro-particle suspension	0.33±0.32	1.13±0.75
	Ultrafine rebamipide suspension	0.91±1.15	2.08±1.18
tmax (h)	Rebamipide micro-particle suspension	1	0.25
	Ultrafine rebamipide suspension	0.25	0.25
t1/2 (h)	Rebamipide micro-particle suspension	5.38	8.19
	Ultrafine rebamipide suspension	5.56	9.04
t1/2 (range)	Rebamipide micro-particle suspension	4–24 h	0.25–24 h
	Ultrafine rebamipide suspension	2–24 h	0.25–24 h
AUCt (µg·h/g)	Rebamipide micro-particle suspension	2.61	14.1
	Ultrafine rebamipide suspension	4.16	16.9
AUCinf (µg·h/g)	Rebamipide micro-particle suspension	2.74	15.5
	Ultrafine rebamipide suspension	4.38	20.2

Rebamipide micro-particle suspension: Commercially available 2% rebamipide micro-particle suspension. Ultrafine rebamipide suspension: 2% ultrafine rebamipide suspension (eye-drop formulation). After single administration into both eyes of 4 animals at a dose of 50 µL/eye, the cornea and conjunctiva were harvested from individual 4 animals (8 eyes) at each time point (0.25, 1, 2, 4, 8, and 24 h). The corneal and conjunctival concentrations of rebamipide were determined by LC-MS/MS. Parameters for these tissues were calculated from the mean data of 7–8 eyes from 4 animals. Values of C_max represent the mean±standard deviation (S.D.) (n=7–8 eyes, 4 animals).

DISCUSSION

A commercially available 2% rebamipide micro-particle white milky suspension causes a short interval of blurring in a patient’s vision after ocular administration.⁹⁾ In the present study, we prepared a novel aqueous suspension of ultrafine rebamipide with a higher level of transparency (Fig. 6) and a level of efficacy similar to that of the commercially available rebamipide micro-particle suspension (Fig. 8) for ophthalmic use. The eye-drop formulation we obtained would prevent the momentary blurring of a patient’s vision. The eye drops require no redispersion prior to placement in the eye, because it forms no visible particle sedimentation during storage at room temperature for up to 3 years (Fig. 7). These features of the ultrafine rebamipide suspension can greatly improve convenience and compliance for patients. In addition, the eye-drop formulation composed of ultrafine rebamipide nanoparticles is easily prepared at little additional cost, is easy to scale up, and enables filtration sterilization with a membrane filter (pore size: 0.2 µm). These features of the ultrafine rebamipide suspension also give it great potential from the viewpoint of industrial manufacturing.

To obtain a highly transparent ultrafine rebamipide suspension, we at first choose the wet beads milling. However, it still produced white milky suspensions, although mean particle sizes were less than 300 nm. As shown in Table 1, since small primary particle of rebamipide were prepared by the neutralization crystallization without any additive (Table 1), we then choose the neutralizing crystallization method^18,19) with several improvements to obtain a rebamipide suspension with high transparency and then recognized that the following four points were critical: selection of an additive at the point of crystallization (Step 1), establishing the process parameters at crystallization (Step 2), use of the dispersion method (Step 3), and dialysis (Step 4). As summarized in Table 1, at the point of crystallization, the type and molecular weight of the additives obviously affected the particle size, particle aggregation, and transparency. On the basis of providing a higher transmittance following dispersion, a HPMC (TC-5) with a low molecular weight was selected as an additive for the final eye-drop formulation. Then, optimization of the process parameters at the point of crystallization enhanced the transparency of the suspension, although the optimization did not change the particle size (Table 3). This suggests that the shearing speed at crystallization might affect only the particle shape, resulting in an improvement in transparency. In order to disperse the particle aggregation of crystalized suspensions (Step 3), a liquid–liquid shearing method was adopted, since other dispersion methods such as the high-speed rotary homogenizer (Table 1), the wet beads milling, a high pressure homogenizer and a nanomizer could not decrease the particle size and they could not increase the transparency notably (data not shown). The liquid–liquid shearing method is known to generate a very strong liquid–liquid shearing force by counter-rotation with a screen and a rotor at high speed.²³⁾ The strong shearing force derived by the liquid–liquid shearing method can disperse aggregated particles that are incapable of being dispersed by other methods, which results in a higher transparency of the crystalized rebamipide suspensions (Fig. 3). And finally, dialysis with the dispersed suspension (Intermediate-2 after Step-3) prevented particle re-aggregation and gelling as well as an increased transparency of the suspension (Fig. 6, Tables 2, 3).

The transparency of ultrafine rebamipide suspensions containing TC-5 was superior to that of ultrafine rebamipide suspensions containing PVP K25 (Fig. 3), although the particle sizes were the same (approximately 100 nm) (Fig. 3). Observation via transmission electron microscope showed that the crystal shape of ultrafine crystallized rebamipide with 1% PVP K25 after dialysis (Step 4) had the shape of a homogenous needle crystal with long-gage and short-gage lengths of approximately 200 and 40 nm, respectively. On the other hand, the crystal shape of ultrafine crystallized rebamipide with 1% TC-5 after dialysis was had a homogenous hyper-needle crystal shape but with a long-gage length of 300 to 1000 nm and a short-gage length of about 15 nm (Fig. 4). Less scattering for visible light is required before a suspension can be transparent.²⁴⁾ Light scattering by ultrafine particles that are smaller than the wavelength of visible light (380–780 nm) is mainly a result of Rayleigh scattering. Ultrafine particles that are smaller than 1/4 of the wavelength of visible light (95–195 nm) reduce light scattering and drastically enhance transparency.²⁵⁾ However, at higher concentrations (such as 2%), ultrafine suspensions with particle sizes of 100 nm are generally opaque because the suspensions contain larger particles within their size distribution. In the present study, the 2% rebamipide ultrafine suspension with TC-5 showed an improvement in the transparency of a rebamipide suspension (Fig. 6). This could have been due to the homogenous hyper-needle crystal shape of the prepared ultrafine particles of rebamipide, which could have suppressed the scattering of visible light. Meanwhile, the X-ray diffraction spectra indicated that ultrafine rebamipide crystals with TC-5 or PVP K25 maintained the same crystal form as the bulk rebamipide powder (Fig. 5). It was interesting that the co-existence of polymers at the point of crystallization did not affect the crystal form, but did affect the crystal morphology.

Kassem et al. reported that the nano-suspensions enhanced the rate and extent of the ophthalmic drug absorption as well as the intensity of drug activity by comparison with the effect of the micro-crystalline suspensions.²⁶⁾ In the present study, the rebamipide ultrafine suspension also offered a slightly higher distribution of rebamipide in the ocular tissues compared with that of the micro-particle suspension. Atomization of a drug with low solubility might increase the dissolution rate owing to an enlargement of the drug’s specific surface.^12,13) With respect to micro-particle suspensions, undissolved residual rebamipide on the ocular surface after ocular administration may be discharged from the conjunctival sac owing to a slower dissolution rate, whereas the residual rebamipide of ultrafine particles on the ocular surface may dissolve much faster than those of micro-particles, and result in a slightly higher distribution of rebamipide in the ocular tissues within a short time after administration (Fig. 9). On the other hand, the increases in conjunctival mucin action from a 2% ultrafine rebamipide suspension and that of a 2% micro-particle rebamipide suspension were comparable (Fig. 8). A slightly higher conjunctival distribution from a rebamipide ultrafine suspension might not affect the increases in conjunctival mucin action. Otherwise, the increases in the action of conjunctival mucin for both ultrafine (Fig. 8) and micro-particle⁶⁾ suspensions might have reached the maximal effect with rebamipide concentrations of 2% or less in the rabbits that were tested for the present study.

CONCLUSION

An aqueous suspension of ultrafine rebamipide particles is expected to have a pharmacological effect that is similar to the commercially available micro-particle suspension. The rebamipide suspension, however, would improve patient adherence because of a highly transparent nature that prevents the customary blurring of vision, and also because of easy administration with no need for redispersal.

Acknowledgments

The authors wish to thank Dr. Koji Yamamoto, Otsuka Pharmaceutical Co., Ltd., for his cooperation in the transmission electron microscope measurements. We thank Mr. J. L. McDonald for his helpful advice in preparing this manuscript.

Conflict of Interest

TM, SH, HU, AO are current employees of Otsuka Pharmaceutical Co., Ltd. All work was funded by Otsuka Pharmaceutical Co., Ltd.

REFERENCES

• 1) Uchida M, Tabusa F, Komatsu M, Morita S, Kanbe T, Nakagawa K. Studies on 2(1H)-quinolinone derivatives as gastric antiulcer active agents. 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]propionic acid and related compounds. Chem. Pharm. Bull., 33, 3775–3786 (1985).
• 2) Yamasaki K, Ishiyama H, Imaizumi T, Kanbe T, Yabuuchi Y. Effect of OPC-12759, a novel antiulcer agent, on chronic and acute experimental gastric ulcer, and gastric secretion in rats. Jpn. J. Pharmacol., 49, 441–448 (1989).
• 3) Kinoshita S, Awamura S, Oshiden K, Nakamichi N, Suzuki H, Yokoi N. Rebamipide OPC-12759 in the treatment of dry eye: a randomized, double-masked, multicenter, placebo-controlled phase II study. Ophthalmology, 119, 2471–2478 (2012).
• 4) Kinoshita S, Oshiden K, Awamura S, Suzuki H, Nakamichi N, Yokoi N, Rebamipide Ophthalmic Suspension Phase 3 Study Group. A randomized, multicenter phase 3 study comparing 2% rebamipide (POC-12759) with 0.1% sodium hyaluronate in the treatment of dry eye. Ophthalmology, 120, 1158–1165 (2013).
• 5) Urashima H, Okamoto T, Takeji Y, Shinohara H, Fujisawa S. Rebamipide increases the amount of mucin-like substances on the conjunctiva and cornea in the N-acetylcysteine-treated in vivo model. Cornea, 23, 613–619 (2004).
• 6) Urashima H, Takeji Y, Okamoto T, Fujisawa S, Shinohara H. Rebamipide increases mucin-like substance contents and periodic acid Schiff reagent-positive cells density in normal rabbits. J. Ocul. Pharmacol. Ther., 28, 264–270 (2012).
• 7) Tanaka H, Fukuda K, Ishida W, Harada Y, Sumi T, Fukushima A. Rebamipide increases barrier function and attenuates TNFα-induced barrier disruption and cytokine expression in human corneal epithelial cells. Br. J. Ophthalmol., 97, 912–916 (2013).
• 8) Ueta M, Sotozono C, Yokoi N, Kinoshita S. Rebamipide suppresses PolyI:C-stimulated cytokine production in human conjunctival epithelial cells. J. Ocul. Pharmacol. Ther., 29, 688–693 (2013).
• 9) Otsuka Pharmacutical Co., Ltd., Interview form of Mucosta^® ophthalmic suspension UD2%, 5 (2015).
• 10) Müller RH, Gohla S, Keck CM. State of the Art of Nanocrystals-Special Features, Production, Nanotoxicology Aspects and Intracellular Delivery. Eur. J. Pharm. Biopharm., 78, 1–9 (2011).
• 11) Kondo N, Iwao T, Masuda H, Yamanouchi K, Ishihara Y, Yamada N, Haga T, Ogawa Y, Yokoyama K. Improved oral absorption of a poorly water-soluble drug, HO-221, by wet-bead milling producing particles in submicron region. Chem. Pharm. Bull., 41, 737–740 (1993).
• 12) Liversidge GG, Cundy KC. Particle size reduction for improvement of oral bioavailability of hydrophobic drugs: I. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. Int. J. Pharm., 125, 91–97 (1995).
• 13) Merisko-Liversidge E, Liversidge GG, Cooper ER. Nanosizing: a formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci., 18, 113–120 (2003).
• 14) Sauron R, Wilkins M, Jessent V, Dubois A, Maillot C, Weil A. Absence of food effect with a 145 mg nanoparticle fenofibrate tablet formulation. Int. J. Clin. Pharmacol. Ther., 44, 64–70 (2006).
• 15) Jinno J, Kamada N, Miyake M, Yamada K, Mukai T, Odomi M, Toguchi H, Liversidge GG, Higaki K, Kimura T. Effect of particle size reduction on dissolution and oral absorption of a poorlywater-soluble drug, cilostazol, in beagle dogs. J. Control. Release, 111, 56–64 (2006).
• 16) Hu J, Johnston KP, Williams RO III. Nanoparticle engineering processes for enhancing the dissolution rates of poorly water soluble drugs. Drug Dev. Ind. Pharm., 30, 233–245 (2004).
• 17) Gassmann P, List M, Schweitzer A, Sucker H. Hydrosols—alternatives for the parenteral application of poorly water soluble drugs. Eur. J. Pharm. Biopharm., 40, 64–72 (1994).
• 18) Hirakawa Y, Harada K. Development of methods of ultra-fine size reduction of slightly soluble medicinal crystals (2). Factors affecting the ultra-fine size reduction of oxolinic acid crystals. Yakugaku Zasshi, 103, 690–695 (1983).
• 19) Hirakawa Y, Harada K. Studies on development of the ultrafine size reduction method of slightly soluble medicinal crystals. V. Size reduction of phenytoin and phenobarbital. Yakugaku Zasshi, 104, 91–96 (1984).
• 20) Chan HK, Kwok PCL. Production methods for nanodrug particles using the bottom-up approach. Adv. Drug Deliv. Rev., 63, 406–416 (2011).
• 21) Vehring R. Pharmaceutical particle engineering via spray drying. Pharm. Res., 25, 999–1022 (2008).
• 22) Hasegawa S, Sekino H, Matsuoka O, Saito K, Sekino H, Morikawa A, Uchida K, Koike M, Azuma J. Bioequivalence of Rebamipide Granules and Tablets in Healthy Adult Male Volunteers. Clin. Drug Investig., 23, 771–779 (2003).
• 23) Hisamitsu I, Hagihara T. The nano-sized emulsion by the big share stress by jet-flow method. Fragrance Journal, 31, 69–73 (2003).
• 24) Van de Hulst HC. Light Scattering by Small Particles. Wiley, New York, NY (1957).
• 25) Saitoh M. Properties and Applications of Ultra fine Ceramic Powders. Powder science & engineering, 30, 35–40 (1998)
• 26) Kassem MA, Abdel Rahman AA, Ghorab MM, Ahmed MB, Khalil RM. Nanosuspension as an ophthalmic delivery system for certain glucocorticoid drugs. Int. J. Pharm., 340, 126–133 (2007).