Enhancing Corneal Drug Penetration Using Penetratin for Ophthalmic Suspensions

Abstract

Eye drops, including solutions and suspensions, are essential dosage forms to treat ophthalmic diseases, with poorly water-soluble drugs typically formulated as ophthalmic suspensions. In addition to low bioavailability, suspensions exhibit limited efficacy, safety, and usability due to the presence of drug particles. Improving bioavailability can reduce the drug concentrations and the risk of problems associated with suspended drug particles. However, practical penetration enhancers capable of improving bioavailability remain elusive. Herein, we focused on penetratin (PNT), a cell-penetrating peptide (CPP) that promotes active cellular transport related to macromolecule uptake, such as micropinocytosis. According to the in vitro corneal uptake study using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24, PNT enhanced the uptake of Fluoresbrite^® YG carboxylate polystyrene microspheres without covalent binding. In an ex vivo porcine eye model, the addition of 10 µM PNT to rebamipide ophthalmic suspension markedly improved the corneal uptake of rebamipide; however, the addition of 100 µM PNT was ineffective due to potentially increased particle size by aggregation. This article provides basic information on the application of PNT as a penetration enhancer in ophthalmic suspensions, including the in vitro and ex vivo studies mentioned above, as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay and storage stability at different pH values.

INTRODUCTION

Among the five human senses, vision accounts for the highest percentage of perception. A survey of the senses most valued by the general public in the U.K. suggested that sight is the most valued sense, followed by hearing.¹⁾ Visual loss also causes the most substantial reduction in QOL and is associated with depression, indicating the importance of vision in QOL.²⁾ Preventing visual loss due to ophthalmologic diseases is crucial to maintaining QOL, and it is desirable to offer as many effective medications as possible to the market to address unmet medical needs.

Eye diseases can be divided into four major categories: visual dysfunction, extraocular, intraocular, and fundus diseases. Visual dysfunctions include refractive error, strabismus, dysregulation, presbyopia, and eyestrain, whereas extraocular diseases include keratoconjunctival epithelial disorders, ocular infections, and ocular allergies. Intraocular diseases include glaucoma, cataracts, endophthalmitis, and vitreous opacity, whereas fundus diseases include retinopathy, age-related macular degeneration, retinal detachment, and optic neuritis. Topical administration of eye drops, which account for approximately 90% of the ophthalmic drug market,³⁾ has been used to treat these diseases. However, drug delivery to posterior ocular tissues is difficult owing to challenges such as low bioavailability caused by tear fluid turnover, elimination via the nasolacrimal duct, blinking eyes, and physicochemical biological barriers such as the cornea.⁴⁾ Thus, several intravitreal injectable solutions have been approved and clinically used to treat endophthalmitis and fundus diseases. These injectable solutions enable efficient drug delivery to the eye with high bioavailability; however, they are highly invasive.

Although penetration enhancers for ocular drug delivery have long been explored to improve bioavailability, practical penetration enhancers remain elusive, and safer and more effective penetration enhancers need to be developed. Previously, we have shown that cell-penetrating peptides (CPPs) can improve the corneal uptake of drug molecules, even when the drug molecules coexist with CPPs without conjugation. Furthermore, the improvement potentially correlated with the interaction between CPPs and drug molecules, as quantified by surface plasmon resonance.⁵⁾ Similarly, Kamei et al. and Khafagy et al. have reported that intestinal and nasal absorption of insulin is enhanced by CPPs. This enhancement was also observed under conditions where insulin coexisted with CPPs without conjugation. Furthermore, the effect of this enhancement correlated with the interaction of insulin and CPPs.^6,7) Subsequently, we screened representative CPPs that have demonstrated efficacy as penetration enhancers in other routes of administration. Among these, penetratin (PNT) emerged as the most potent enhancer for corneal drug delivery.⁵⁾ The advantage of PNT is that it can work as a safe corneal penetration enhancer even when coexisting with drugs. However, PNT also has limitations: drugs need to interact with PNT to obtain the enhancement via PNT. Additionally, the enhancement via PNT on suspended drug particles that are not dissolved in the formulation remains unclear.

As mentioned above, most ophthalmic formulations available in the market are solutions, with suspensions accounting for only a small percentage. Suspensions are thermodynamically unstable systems and exhibit complex quality issues, such as uniformity, aggregation, sedimentation of suspended particles, resuspension, and manufacturability, which differ from those of solutions.⁸⁾ Formulation optimization, including particle size control and improving uniformity by adding water-soluble polymeric excipients, can help address these quality issues and facilitate proper administration according to the dosage regimen and adequate bioavailability.

Generally, a smaller particle size can enhance C_max and shorten T_max in the aqueous humor and cornea.⁹⁾ Formulation viscosity may also affect bioavailability and ensure formulation uniformity and resuspension. Previous reports have shown that bioavailability may change depending on the particle size and viscosity,¹⁰⁾ suggesting that particle size and viscosity are critical quality attributes for controlling usability, efficacy, and safety.

Thus, successful development of ophthalmic suspensions involves many complex factors associated with efficacy, safety, and usability. Identifying penetration enhancers that can improve the corneal uptake of suspended particles will help mitigate various risks.

This study aimed to investigate the potential of PNT to improve the corneal penetration of suspended particles. The effect of particle size on the improvement by PNT was evaluated using polystyrene microspheres (PM) of different particle sizes, and the mechanism of improvement was evaluated using inhibitors of active cellular transport pathways. The potential application of PNT in rebamipide ophthalmic suspensions was then evaluated based on the effects of the amount of PNT added on the physicochemical properties and uptake into the cornea and aqueous humor. Another task was to evaluate the storage stability to determine the stability risks of PNT as the most important quality attribute, and a stability prediction analysis was performed using Arrhenius plots.

MATERIALS AND METHODS

Synthesis of Penetratin

PNT was synthesized using solid-phase peptide synthesis,¹¹⁾ and identified using an LC/MS (LCMS2020, Shimadzu Corporation, Kyoto, Japan), which supported the PNT structure. All reagents were commercially available.

In Vitro Corneal Uptake Study

Different particle sizes (0.05, 0.2, and 0.5 µm) of Fluoresbrite^® YG carboxylate polystyrene microspheres (PM-COOH) and YG polystyrene microspheres (PM-plain; Polysciences Inc., PA, U.S.A.) were evaluated as model suspension particles. The suspension was diluted in phosphate-buffered saline (PBS) to prepare test samples, including 0.01%PM and 10 µM PNT. Following exposure of the 50 µL sample to LabCyte CORNEA-MODEL24 (Japan Tissue Engineering Co., Ltd., Aichi, Japan) (LabCyte), incubation was performed in a CO₂ incubator (MCO-345, SANYO, Osaka, Japan) for a specified time. The samples were completely washed from the surface of LabCyte with PBS, and 50 µL of PBS was added to the surface to prevent drying. Fluorescence intensity was determined using ImageJ (National Institutes of Health, Bethesda, MD, U.S.A.), and fluorescence images were captured using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Cytotoxicity Assay

After the corneal uptake study, LabCyte was placed on well plates with 0.5 mL of 0.5 mg/mL MTT diluted in PBS, followed by incubation in a CO₂ incubator for 3 h and immersion in 300 µL of 2-propanol to extract insoluble formazan dye. Cell viability was calculated based on absorbance at 540–655 nm measured using a microplate reader (model 3550, BIO-RAD Laboratories, CA, U.S.A.), and the positive control (PBS group) was adjusted to 100%.

In Vitro Study Using Endocytosis Inhibitors and at Refrigerated Condition for Mechanism Analysis

To evaluate the involvement of endocytosis, an in vitro corneal uptake study was performed at 4 °C as a condition to inhibit endocytosis.¹²⁾ In addition, two inhibitors were used: Dynasore hydrate (Sigma-Aldrich. St. Louis, MO, U.S.A.), which is a GTPase inhibitor targeting dynamin-1, dynamin-2, and Drp1 (from mitochondria) as an inhibitor of clathrin- and caveolae-mediated endocytosis,¹³⁾ and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) (Sigma-Aldrich), which is an amiloride derivative of sodium/proton exchanger inhibitor as a macropinocytosis inhibitor,¹⁴⁾ to evaluate the contribution of each active transport mechanism.

Ex Vivo Study Using Porcine Eye

Commercially available Rebamipide ophthalmic suspension 2% (Santen Pharmaceutical Co., Ltd., Osaka, Japan) was used for this study: the suspension without PNT as a control sample and the suspension with 10 or 100 µM of PNT dissolved as test samples. The samples were characterized by appearance (digital microscope VHX-7000, KEYENCE, Osaka, Japan), particle size distribution, zeta potential (ZetaSizer NanoZS, Malvern Panalytical Ltd., U.K.), pH (LAQUAtwin, HORIBA Advanced Techno, Co., Ltd., Kyoto, Japan), and osmolality (OM-6060, Arkray Inc., Kyoto, Japan).

Porcine eyes (Osaka-City General Meat Market Inc., Osaka, Japan) were removed from the slaughtered pigs on the morning of the study day. The eyeballs were trimmed and placed in a 12-well plate filled with 1 mL of Neurobasal™-A medium (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, U.S.A.), with the cornea at the top (Fig. 1A). PluriStrainer (pluriSelect Life Science UG, haftungsbeschränkt & Co., KG), with its mesh section removed, was attached to the well to immobilize the eyeball and hold the test formulation on the ocular surface. Subsequently, 1.4 mL of the test formulation was applied (Fig. 1B), and it was confirmed that the test formulation had completely covered the entire ocular surface. The wells were incubated in a CO₂ incubator at 37 °C with parafilm to prevent evaporation. After 1, 2, and 4 h, the corneal surface was thoroughly washed with PBS, and the aqueous humor was collected using a 1 mL Terumo syringe with a 27G needle (Terumo Corporation, Tokyo, Japan). The cornea was trimmed and homogenized (8200 rpm, 2 × 15 s) with 0.6 mL saline using a bead homogenizer (Predellas Evolution, Bertin Technologies SAS, France), followed by adjustment to 5 mL with N,N-dimethylformamide (DMF). After 30 min, the sample was centrifuged (3000 rpm, 25 °C, 10 min), and the supernatant was collected and diluted in an equal volume with a mixture of 20 mM phosphate buffer (pH 6.4) and acetonitrile (83/17). The rebamipide content in the aqueous humor and cornea was determined using ultra-performance liquid chromatography (UPLC) (ACQUITY UPLC H-Class, Nihon Waters, Tokyo, Japan). The other cornea was trimmed into φ4 mm × 3 pieces to perform the MTT cell viability assay (Fig. 1C). All experimental procedures were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and the Animal Care and Use Committee at Santen Pharmaceutical Co., Ltd.

Fig. 1. Image of ex Vivo Study Using Porcine Eye

The eyeball is inserted into a 12-well plate filled with 1.0 mL of Neurobasal™-A medium (A). Subsequently, 1.4 mL of the test sample is applied to the corneal surface, covering the entire surface of the eye (B). After the study, the cornea is trimmed to φ4 mm ×3 pieces for the MTT cell viability assay (C).

Stability Study of PNT

PNT (44.5 µM) was dissolved in diluted MacIlvein buffered saline adjusted to pH 5, 7, and 9, which was then used to evaluate the storage stability of PNT in aqueous solutions at different pH values at 40, 25, and 5 °C. The PNT content was determined by UPLC, with the residual content calculated relative to the initial content. From these residual contents, the long-term stability of PNT at 5 and 25 °C at each pH was predicted from an Arrhenius plot.

RESULTS

Effect of Particle Size and Terminal Group of Drug Particles on Corneal Uptake

Evaluating the effect of PNT on improved corneal uptake of model suspended particles, we found no improvement in PM-plain uptake (Figs. 2A, B). Conversely, PM-COOH uptake was improved, and differences were also observed between the particle sizes (Figs. 3A, B). Cell viability remained unchanged (Figs. 4A, B), suggesting that PNT can safely improve corneal uptake of suspended particles with a negative charge and 0.05–0.5 µm particle size.

Fig. 2. Effect of PNT on Corneal Uptake of PM 2 h after Dosing PM-Plain with or without PNT (A)

Each bar represents the mean ± standard deviation (S.D.), n = 3. ns indicates that the difference is not significant. w/o PNT refers to the sample without PNT. The fluorescence imaging shows LabCyte after in vitro corneal uptake. The exposure time is 1/1.5 ms and the scale bar corresponds to 200 µm (B).

Fig. 3. Effect of PNT on Corneal Uptake of PM 2 h after Dosing PM-COOH with or without PNT (A)

Each bar represents the mean ± S.D., n = 3. The symbol ** indicates p < 0.01 and the symbol * indicates p < 0.05 by t-test. w/o PNT refers to the sample without PNT. The fluorescence imaging shows LabCyte after in vitro corneal uptake. The exposure time is 1/1.5 ms and the scale bar corresponds to 200 µm (B).

Fig. 4. MTT Assay of Corneal Epithelial Cells after in Vitro Corneal Uptake with or without PNT

Each bar represents the mean ± S.D., n = 3. ns indicates that the difference is not significant. w/o PNT refers to the sample without PNT. The cell viability of PM-plain (A) and PM-COOH (B) with PNT is calculated relative to the group without PNT.

In Vitro Study Using Endocytosis Inhibitors and at Refrigerated Condition for Mechanism Analysis

Although the study at 37 °C showed a difference between with and without PNT in 0.5 µm PM-COOH uptake, the difference between with and without PNT disappeared at 4 °C (Fig. 5), suggesting that the improvement by PNT was potentially involved in cellular active transport pathways. Furthermore, the improved uptake in 0.05 and 0.5 µm PM-COOH was no longer observed with the addition of clathrin- and caveolae-mediated endocytosis inhibitors (Fig. 6). Cell viability remained unaltered in these experiments, confirming that the analysis was performed on viable cells (Fig. 7).

Fig. 5. Mechanistic Analysis of Corneal Uptake of 0.5 µm PM by PNT under 4 °C Condition

The fluorescence intensity of 0.5 µm PM without PNT at 37 °C serves as the standard for comparison. Each bar represents the mean ± S.D., n = 3. The symbol * indicates p < 0.05 and the symbol ^†† indicates p < 0.01 by t-test. ns indicates that the difference is not significant. w/o PNT refers to the sample without PNT.

Fig. 6. Mechanistic Analysis of Corneal Uptake of 0.5 and 0.05 µm PM by PNT with Endocytosis Inhibitors

The fluorescence intensity of 0.5 and 0.05 µm PM without PNT serves as the standard for comparison. Each bar represents the mean ± S.D., n = 3. The symbol * and ^† indicate p < 0.05 by t-test versus each result without PNT and inhibitors.

Fig. 7. MTT Assay of Corneal Epithelium Cell Following in Vitro Corneal Uptake for Mechanistic Analysis Using 0.05 and 0.5 µm PM-COOH with PNT, Dynasore, EIPA and under 4 °C Condition

Each bar represents the mean ± S.D., n = 3. ns indicates that the difference is not significant.

Effect of PNT on the Corneal Penetration of Rebamipide in ex Vivo Study Using Porcine Eyes

To confirm the application of PNT to enhance the penetration of active suspended drugs, rebamipide was evaluated as a model drug (Fig. 8). Based on sample characterization, the samples showed no difference in the particle size distribution between the 10 µM PNT and without PNT groups, and the zeta potential shifted to the positive side. Microscopic images of the 100 µM PNT group suggested aggregation, likely attributed to larger particle size distribution (Fig. 9). These suspensions were applied to porcine eyes, and the amount of rebamipide transferred to the cornea and aqueous humor was determined. The corneal rebamipide concentration improved in the 10 µM PNT group, and that in the 100 µM PNT group was equal to or less than that in the no-PNT group. The rebamipide concentration in the aqueous humor showed the same trend as that in the cornea at 2 h; however, the difference disappeared at 4 h (Fig. 10). After 2 h, the corneal cell viability remained unchanged in both groups (Fig. 11).

Fig. 8. Chemical Structure of Rebamipide

Fig. 9. Physicochemical Property of Commercially Available Rebamipide Ophthalmic Suspension 2% (Santen) with or without PNT

Particle size distribution and zeta potential were measured by Zetasizer NanoZS (Malvern). The appearance was observed using a digital microscope VHX-7000 (KEYENCE). pH was measured by a pH meter LAQUAtwin (HORIBA). Osmolality was measured by osmostation OM-6060 (Arkray).

Fig. 10. Effect of PNT Concentration on Corneal Uptake of Rebamipide

The concentration of rebamipide in the cornea (A) and aqueous humor (B) was measured over time in the porcine eye (mean ± S.E.M., n = 3). The study used commercially available rebamipide ophthalmic suspension 2% without PNT as a control sample and the suspension with 10 or 100 µM PNT dissolved as test samples. w/o PNT refers to the control sample without PNT.

Fig. 11. Porcine Corneal Cell Viability by MTT Assay after 2 h Incubation with or without PNT. Each Bar Represents the Mean ± S.E.M., n = 6

Cell viability was calculated relative to the PBS control. ns indicates that the difference is not significant. The test samples include commercially available rebamipide ophthalmic suspension 2% without PNT and the suspension with 10 or 100 µM PNT dissolved.

Effect of pH on Storage Stability of Penetratin

The stability of PNT was evaluated in buffer solutions with distinct pH values; the PNT content decreased with increasing storage temperatures and pH values (Fig. 12). After storage, the pH decreased slightly at pH 9 owing to the low buffering capacity at pH 9; however, the change was within the expected range for evaluating the effect of pH on stability (Table 1). The PNT content was determined to follow the Arrhenius plot because the linear correlation between the logarithm of the reaction rate constant and the reciprocal of the temperature at each pH was evaluated, and the R² values were 0.9961, 1.0000, and 0.9425 at pH 5, 7, and 9, respectively. The activation energy and ln A were calculated to predict the percentage of PNT remaining at 25 and 5 °C (Fig. 13). At 5 °C, more than 90% of PNT persisted at pH 5 and 7, although degradation occurred over time at pH 9. However, at 25 °C, PNT decreased gradually under all pH conditions, particularly at pH 9, which was predicted to almost disappear within one year.

Fig. 12. Remaining PNT after Storage at Different Temperature

The remaining PNT content was calculated relative to the initial PNT content at 40 °C (A), 25 °C (B), and 5 °C (C). All data represent the average of n = 2 measurements.

Table 1. pH of the Samples after Storage

			5 °C		25 °C		40 °C
	Initial	1 month	2 month	2 week	4 week	2 week	4 week
pH 5	4.99	4.96	4.94	4.93	5.07	4.76	4.80
pH 7	6.92	6.96	6.91	6.92	6.88	6.80	6.82
pH 9	8.78	8.75	8.76	8.01	7.96	7.89	7.90

Fig. 13. Remaining of PNT Simulated by Arrhenius Plot at 25 °C (A) and 5 °C (B)

This simulated data was calculated from the actual data shown in Fig. 12.

DISCUSSION

As the application of CPPs for ocular topical administration, peptides to facilitate ocular delivery and TAT peptides have been shown to enhance efficient drug delivery to several ocular tissues, including the corneal epithelium.¹⁵⁾ Considering these techniques, the drug and CPP form a covalently bound conjugate that differs from the original drugs, demonstrating that challenges persist in the application of these techniques to approved drugs with clinical proof-of-concept owing to the requirement to demonstrate efficacy and safety as a novel drug.

Most poorly soluble drugs—approximately 40% of approved drugs and 90% of drug candidates in the discovery phase¹⁶⁾—need to be developed as ophthalmic suspensions with low bioavailability for topical ocular administration. However, simple methods to improve the bioavailability of suspended drugs remain unclear. In the current study, we investigated the potential of PNT to improve corneal uptake of suspended particles with zero dissolved fractions using PM. To evaluate the effect of electrostatic interactions between suspended particles and PNT, carboxy-group-modified PM-COOH was performed, in addition to regular PM-plain; the zeta potentials of PM-plain and PM-COOH are reportedly −15 and −78 mV, respectively.¹⁷⁾ PM was found to be actively incorporated through cell phagocytosis (part of endocytosis) in LM fibroblasts, which is a transformed cell line with high endocytic capacity,¹⁸⁾ and thus, we expect that corneal active transport can enhance take up undissolved particles.

Although PNT minimally impacts PM-plain uptake, the uptake of PM-COOH was improved (Figs. 2A, B, 3A, B), thereby suggesting that electrostatic interactions between PNT and PM-COOH may have improved PM-COOH uptake along with cellular uptake of PNT. Given that cell viability remained unchanged (Figs. 4A, B), we considered the possibility that the cellular active transport pathways are maintained and involved in uptake.

Endocytosis is known as one of active transport pathways, including clathrin- and caveolae-mediated endocytosis, in which the size of vesicles depends on the size of the substances being migrated.^19–21) Accordingly, they may have been involved in different degrees of contribution to the uptake of small particles of 0.5 µm or less and may have been activated by PNT.

The intracellular translocation mechanism of PNT reportedly involves direct translocation and endocytosis,²²⁾ and direct translocation is dominant at low concentrations below 2 µM, whereas both endocytosis and direct translocation occur at high concentrations.²³⁾ Dom et al. analyzed the cell migration of DNA by PNT using COS7 cells (monkey kidney fibroblast cell line) and found that PNT could improve uptake at both 37 and 4 °C, involving hydrophobic and electrostatic interaction with cells and destabilization of the lipid bilayer membrane. The authors also suggested that uptake was reduced because of impaired membrane destabilization in W6F-PNT, in which the sixth tryptophan of PNT was replaced with phenylalanine.²⁴⁾ Such findings have been reported in other fibroblasts, such as L929 cells (mouse fibroblast cell line), SV80 cells (human lung fibroblast cell line), and HeLa cells (epithelial cells),²⁵⁾ suggesting that both direct translocation and endocytosis play a role in improving the uptake of particles through PNT.

In our study, comparing the fluorescence intensity of without PNT at 37  and 4 °C shows a significant change in fluorescence intensity, which is likely due to the reduced uptake of particles at 4 °C (Fig. 5), where the active transport pathways are mostly halted. Additionally, clathrin- and caveolae-mediated endocytosis inhibitors and macropinocytosis inhibitors attenuated the intensity (Fig. 6), suggesting that the active transport pathways appear to be dominant and direct translocation is rarely involved in the improved corneal uptake of suspended drugs by PNT. This finding aligns with a report by Nagai et al., which demonstrated the role of endocytosis in the transcorneal penetration of indomethacin nanoparticle formulations without CPPs.²⁶⁾

As far as our knowledge, we have not seen any reports describing the penetration enhancement of corneal penetration of small molecule drugs through PNT without covalent binding, as observed in this study. Some studies on the intestinal absorption of peptides and proteins, such as insulin and GLP-1, have reported that electrostatic interactions between CPPs and drugs could play important role in enhanced absorption,^27–30) suggesting that pH adjustment of formulations may be a critical formulation attributes.

Regarding the transport mechanism of PNT, factors such as the amino acid sequence, the number of arginine residues, and the secondary structure could affect the mechanism itself.^31,32) However, the impact of formulation attributes on the transport mechanism of PNT itself remains unclear. Additionally, the contribution of direct translocation and active transport pathways, such as endocytosis, may vary depending on the target tissue. Further research is needed to optimize formulations by well understanding the effects of formulation attributes and quality attributes of PNT.

Typically, drug absorption from the ocular surface after eye drop instillation involves distribution phenomena on the ocular surface, which reportedly correlates with the drug log D_7.4 values.³³⁾ Accordingly, undissolved drug particles are unlikely to be absorbed by the ocular surface via passive transport. Although the direct absorption of suspended drugs into the cornea remains unclear, the absorption into the skin, which has a similar mechanism of drug absorption as the cornea, has been reported.³⁴⁾ Polystyrene microspheres barely permeate intact skin and localize at the needle puncture site and pores, but fluorescein isothiocyanate (FITC)-dextran can permeate, indicating the contribution of the dissolved fraction to skin permeation by passive transport.³⁵⁾ The contribution of active transport is considered small, given that the stratum corneum comprises corneocyte without nuclei and cytoplasmic organelles that have already undergone apoptosis and intracellular lipid complex.³⁶⁾ Conversely, active transport may be effective in the corneal epithelium, suggesting that PNT can improve the uptake of suspended particles. Improving uptake via active transport enables the reduction in the concentration of suspended drug particles in ophthalmic suspensions, potentially increasing formulation design options to ensure efficacy and safety, as well as decreasing usability risks due to suspended particles.

These findings highlighted the feasibility of rebamipide ophthalmic suspensions as a model suspension. Rebamipide is commonly used to treat dry eye disease (DED),^37,38) with the formulation typically leaving room for improvement in terms of suspension-specific usability. For example, some side effects, such as short-term blurred vision, feeling of a foreign matter, eye discomfort, and bitterness are reported.³⁹⁾ To improve the short-term blurred vision, some studies have investigated the use of ultrafine rebamipide suspensions.⁴⁰⁾

Improving the corneal uptake of rebamipide through PNT could reduce the concentration of rebamipide in the suspension and provide a value-added product that may reduce the risk of the suspension-specific side effects as described above. As rebamipide possesses a carboxyl group, electrostatic interactions with the cationic amino acids of PNT may contribute to the enhancement in corneal uptake of rebamipide. In an ex vivo study using porcine eyes, corneal uptake of rebamipide was enhanced in the 10 µM PNT group, suggesting that this enhancement participated in the active transport pathways. In contrast, corneal uptake in the 100 µM PNT group was similar to that in the 0 µM PNT group (Fig. 10), suggesting that the increase in the particle size in 100 µM PNT group (Fig. 9) may have reduced the contribution of endocytosis by PNT, and the contribution of passive diffusion of slightly dissolved rebamipide appeared. Although the findings with polystyrene microspheres may explain the mechanism of enhanced corneal uptake of rebamipide particles by PNT, further studies are needed to clearly elucidate the mechanism.

These findings reveal that particle size control is necessary to improve the corneal uptake of undissolved suspended particles with PNT. In addition to particle size control, the current study investigated the storage stability of PNT as the highest risk in designing eye drop formulations, given the characteristics of the peptide modality.

Herein, we evaluated pH stability at pH 5, 7, and 9 and predicted long-term storage stability using an Arrhenius plot (Figs. 12, 13). The sample at pH 9 could not maintain PNT stability even under refrigerated conditions, limiting drug application with an optimal pH above pH 7. Samples with pH values of 5 and 7 were stable under refrigerated conditions, although the PNT content gradually decreased at 25 °C. Kristensen et al. reported that PNT was most stable at pH 5, 6.5, and 7.4 and decreased with increasing pH in short-term stability using a mixture with Caco-2 cells.³⁰⁾ The pI of PNT was found to be 12.31,⁴¹⁾ suggesting that a lower pH leads to higher stability in water owing to the higher net positive charge and lower intermolecular interaction between PNT.

The decrease in PNT content at 25 °C may be resolved by limiting the storage time at room temperature, such as by transporting the products under refrigeration.⁴²⁾ In the current study, the optimal pH range for PNT stability and the pH 5.5–6.5 of rebamipide suspension tend to overlap, suggesting that the storage stability of PNT can be sufficiently ensured.

CONCLUSION

In vitro studies using LabCyte revealed that PNT can improve the corneal uptake of suspended drug particles in ophthalmic suspensions, suggesting that two important factors need to be considered: (1) drug charge and (2) particle size of the suspended drugs. The electrostatic interaction of PNT with negatively charged drug particles may improve corneal uptake, and endocytosis may be involved in the mechanism of corneal intracellular transport. Particle size control is important for corneal uptake via endocytosis, and the optimal amount of PNT should be confirmed to account for the aggregation caused by the addition of PNT. Our findings highlight the potential of PNT as a penetration enhancer in ophthalmic suspensions. We expect that these findings will provide fundamental information to improve the bioavailability of suspended particles and potentially address issues critical to the development of ophthalmic suspensions with improved efficacy, safety, and usability.

Acknowledgments

The authors thank Kazuhito Yamada, Head of Pharmaceutics and Pharmacology Department, Santen Pharmaceutical Co., Ltd., for his cooperation in this collaborative research activity.

Conflict of Interest

R. Morofuji, K. Kudo, and K. Okabe are current employees of Santen Pharmaceutical Co., Ltd. All work was performed at the Joint Laboratory of Nara Institute of Science and Technology and Santen.

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