Diquafosol Ophthalmic Solution Increases Pre- and Postlens Tear Film During Contact Lens Wear in Rabbit Eyes.

OBJECTIVES: To investigate the behavior of prelens tear film (PLTF) and postlens tear film (PoLTF) after the instillation of diquafosol using an experimental rabbit model of eyes with contact lens. METHODS: Cross-sectional, anterior segment optical coherence tomographic images of the inferior midperipheral cornea were obtained at baseline and at 5, 15, 30, 60, 90, and 120 min after the instillation of 3% diquafosol ophthalmic solution in 10 Japanese white rabbits wearing contact lenses. From the obtained images, the areas of the PLTF and PoLTF were calculated. Both artificial tear solution and 0.1% sodium hyaluronate ophthalmic solution were used for comparison. RESULTS: Significant fluid accumulation in both the PLTF and PoLTF was observed after diquafosol instillation, whereas no fluid accumulation was visible after the instillation of artificial tear or sodium hyaluronate. The increase in PLTF area after diquafosol instillation was significantly higher (P<0.01) at 15 and 30 min than that after the instillation of artificial tear or sodium hyaluronate. The increase in PoLTF area up to 60 min after the instillation of diquafosol was significantly higher (P<0.01) than that after the instillation of either of the other two drugs. CONCLUSIONS: Instillation of 3% diquafosol ophthalmic solution increases PLTF and PoLTF in rabbit eyes with contact lenses. Diquafosol has potential as a treatment option for contact lens-related dry eye.

Spontaneous nephroblastoma with oncocytic differentiation in a Japanese White rabbit.

Spontaneous nephroblastoma is an uncommon tumor in laboratory rabbits. We recently encountered this tumor, and we describe its histological characteristics in this report. A male 3-year-old Japanese White rabbit (JW/kbs), maintained as a stock animal, suddenly showed poor condition and was found dead a few days later. At necropsy, a large mass was found that extended from one side of the renal pelvis. The cut surface of the mass was dark red in color and velvety to the touch. The kidney on the contralateral side was normal. Microscopically, the tumor mass consisted of biphasic components, which consisted of epithelial (tubular and glomerular) and blastemal (nodular) elements. No sarcomatous proliferation was observed. In addition, some of the tubules were lined by cells with a large amount of eosinophilic cytoplasm. The cells were confirmed as oncocytes by immunohistochemical and electron microscopic examinations. The present case was therefore diagnosed as a nephroblastoma with oncocytic differentiation.

The effects of oral and topical corticosteroid in rabbit corneas.

BACKGROUND: To determine the most effective route of administration of corticosteroids in the treatment of ocular surface disease, by characterizing the difference between oral prednisolone and topical dexamethasone administration using an animal model. METHODS: Pharmacokinetic analyses determined the corticosteroid concentrations in the normal ocular tissues of rabbits after oral or topical administration of corticosteroids using LC-MS/MS. In wound healing analyses, the area of the epithelial defect created by keratectomy using a 6-mm trephine was calculated with an image analyzer using an orally or topically steroid-administrated animal model. The average size of basal epithelial cells, the frequency of mitotic basal epithelial cells, the number of squamous cells, and the number of hypertrophic stromal fibroblasts were determined in the enucleated corneal tissues after wound closure. RESULTS: By slit lamp examination, no remarkable differences were observed between orally and topically administered groups. Pharmacokinetic analyses showed that the distribution of dexamethasone after topical administration was superior to that after oral administration in the cornea. In contrast, both concentrations of corticosteroid applied topically and orally were similar with regards to AUCs (area under the concentration-time curve) in the conjunctiva. Although the healing rate was slower in the topical group, all corneas were almost healed within 96 h in the wound healing analysis. According to the histological analyses of epithelial cells, the average basal cell size was larger, the frequency of mitotic basal cells was greater, and the number of squamous epithelial cell layers was lower in the topically administered group although all of these differences were with no statistical significance. However, the number of hypertrophic stromal fibroblasts in the topically administered group was significantly lower than that in the orally administered group. CONCLUSIONS: There are different distributions and effects between orally and topically administered corticosteroids on the ocular surface. The data may provide the useful information in selecting the appropriate route of corticosteroid application for the treatment of ocular surface disease.

Trans-eyelid distribution of epinastine to the conjunctiva following eyelid application in rabbits.

PURPOSE: To reveal the penetration of epinastine, an anti-allergic ophthalmic agent, into the eyelid and its distribution to the conjunctiva after administration of a cream formulation on rabbit eyelid skin. STUDY DESIGN: Experimental study. METHODS: Rabbits were treated with 0.5% epinastine cream on hair-shaved eyelids, followed by preparation of eyelid tissue slices to determine spatial tissue distribution of epinastine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification using laser-microdissected tissues and desorption electrospray ionization mass spectrometry imaging (DESI-MSI). In addition, following either eyelid application of 0.5% epinastine cream or ocular instillation of 0.1% epinastine eye drops, concentration-time profiles of epinastine in the palpebral conjunctiva and bulbar conjunctiva were determined using LC-MS/MS. RESULTS: Laser microdissection coupled with LC-MS/MS analysis detected high concentrations of epinastine around the outermost layer of the eyelid at 0.5 h post-administration that gradually diffused deeper into the eyelid and was distributed in the conjunctival layer at 8 and 24 h post-administration. Similar time-dependent drug distribution was observed in high-spatial-resolution images obtained using DESI-MSI. Epinastine concentrations in the conjunctival tissues peaked at 4-8 h after administration of 0.5% epinastine cream and then decreased slowly over 72 h post-administration. In contrast, epinastine concentrations peaked quickly and decreased sharply after epinastine eye drop administration. CONCLUSION: After the application of epinastine cream to the eyelid skin, epinastine gradually permeated the eyelid. The compound was retained in the conjunctiva for 8-24 h post-administration, indicating that epinastine cream is a promising long-acting formulation for treating allergic conjunctivitis.

The Antiglaucoma Agent and EP2 Receptor Agonist Omidenepag Does Not Affect Eyelash Growth in Mice.

Purpose: The present study investigated the effects of the antiglaucoma agent and selective E2 receptor agonist omidenepag isopropyl (OMDI) on eyelash growth in comparison with a prostaglandin analog (prostamide receptor agonist) in mice. Methods: Four-week-old female mice (C57BL/6J) were divided into 3 groups of n = 10 each. The groups were administered 3 µL of 0.003% OMDI solution, the vehicle (negative control), or a 0.03% bimatoprost solution (positive control) on the upper eyelids of the right eyes once daily for 14 days. On the 15th day, all animals were euthanized, and the upper eyelids with eyelashes were fixed with 10% neutral formalin. Eyelashes were evaluated for number, length, and thickness using a stereomicroscope. Specimens were then paraffin-embedded and stained with hematoxylin and eosin, followed by microscopic examination to assess eyelash morphology and growth cycle. Results: Eyelash number (143.5 ± 6.7/eyelid), thickness, and percentage of dermal papilla in the anagen phase in the OMDI group were similar to those observed in the vehicle group (eyelash number, 144.2 ± 5.7/eyelid). In contrast, eyelash number (166.7 ± 7.0/eyelid), thickness, and the percentage of dermal papilla in the anagen phase were significantly greater in the bimatoprost group compared with those of the vehicle group. Conclusions: Unlike existing prostaglandin analogs, our findings indicate that OMDI has no effect on eyelash growth in mice, suggesting that it may be a promising antiglaucoma agent with a reduced number of adverse effects.

MALDI imaging mass spectrometry revealed atropine distribution in the ocular tissues and its transit from anterior to posterior regions in the whole-eye of rabbit after topical administration.

It is essential to elucidate drug distribution in the ocular tissues and drug transit in the eye for ophthalmic pharmaceutical manufacturers. Atropine is a reversible muscarinic receptor used to treat various diseases. However, its distribution in ocular tissues is still incompletely understood. Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) evaluates drug distribution in biological samples. However, there have been few investigations of drug distribution in ocular tissues, including whole-eye segments. In the present study, we explored the spatial distribution of atropine in the whole-eye segment by MALDI-IMS, and then evaluated the changes in atropine level along the anterior-posterior and superior-inferior axes. A 1% atropine solution was administered to a rabbit and after 30 min, its eye was enucleated, sectioned, and analyzed by MALDI-IMS. Atropine accumulated primarily in the tear menisci but was found at substantially lower concentrations in the tissue surrounding the conjunctival sacs. Relative differences in atropine levels between the anterior and posterior regions provided insights into the post-instillation behavior of atropine. Atropine signal intensities differed among corneal layers and between the superior and inferior eyeball regions. Differences in signal intensity among tissues indicated that the drug migrated to the posterior regions via a periocular-scleral route. Line scan analysis elucidated atropine transit from the anterior to the posterior region. This information is useful for atropine delivery in the ocular tissues and indicates that MALDI-IMS is effective for revealing drug distribution in whole-eye sections.

Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips.

PURPOSE: Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA. METHODS: MUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined. RESULTS: MUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%. CONCLUSION: We developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients.

Advantages of Efficacy and Safety of Fixed-Dose Tafluprost/Timolol Combination Over Fixed-Dose Latanoprost/Timolol Combination.

PURPOSE: To compare the safety and efficacy of fixed-dose tafluprost/timolol combination (Taf/T-FDC) with those of fixed-dose latanoprost/timolol combination (Lat/T-FDC) by measuring the intraocular pressure (IOP)-lowering effect, ocular pharmacokinetics, and ocular surface toxicity. METHODS: The IOP-lowering effect of Taf/T-FDC and Lat/T-FDC in ocular normotensive monkeys was evaluated at 4 and 8 h after instillation in study A, at 12, 14, 16, and 18 h after instillation in study B, and at 24, 26, 28, and 30 h after instillation in study C. Drug penetration into the eye was evaluated by measuring the concentrations of timolol, tafluprost acid (active metabolic form of tafluprost), and latanoprost acid (active metabolic form of latanoprost) using liquid chromatography coupled with tandem mass spectrometry after single instillation of Taf/T-FDC or Lat/T-FDC to Sprague Dawley rats. Cytotoxicity following 1-30 min exposure of SV40-transformed human corneal epithelial cells to Taf/T-FDC or Lat/T-FDC was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays. Undiluted and 10-fold diluted solutions of each FDC were evaluated. RESULTS: The IOP-lowering effect of Taf/T-FDC was almost equivalent to that of Lat/T-FDC at 4-8 h after instillation. The peak IOP reduction of Taf/T-FDC and Lat/T-FDC was observed at 8 h after instillation, and there is no difference between the two. The difference between them was observed at 24-30 h after instillation, and Taf/T-FDC demonstrated a significantly greater IOP-lowering effect than Lat/T-FDC at 24-30 h after instillation. The IOP-lowering effect of Taf/T-FDC was sustained up to 30 h after instillation, while that of Lat/T-FDC had almost disappeared at 28 h after instillation. Timolol concentrations in aqueous humor after Taf/T-FDC instillation were higher than those after Lat/T-FDC instillation (Cmax, 3870 ng/mL vs 1330 ng/mL; AUCinf, 3970 ng·h/mL vs 1250 ng·h/mL). The concentrations of tafluprost acid and latanoprost acid in aqueous humor after instillation of Taf/T-FDC and Lat/T-FDC, respectively, were similar to those after instillation of mono-preparations of tafluprost and latanoprost, respectively. The cytotoxic effect of Taf/T-FDC to the human corneal epithelial cells was significantly lower than that of Lat/T-FDC at all evaluated time points in both undiluted and 10-fold diluted FDCs. CONCLUSION: Taf/T-FDC provides increased IOP-lowering effect duration and lower potential ocular surface toxicity than Lat/T-FDC.

Bilateral hamartomatous medullary lipoma within the nasal turbinate bones in a cynomolgus monkey (Macaca fascicularis).

A 15-year-old male cynomolgus monkey (Macaca fascicularis) showed large bilateral masses in the maxillary sinus. In histopathological examination, both masses revealed benign medullary lipomas within the turbinate bones. The tumors were composed of well-developed lipocytes, trabecular bones and a few blood vessels. Although we initially diagnosed the tumor as bilateral lipomas in the nasal turbinates, it was not differentiated from lipomatous hamartoma. Findings, such as unique symmetrical proliferation, lack of border from the normal marrow and the intact surrounding tissue, indicated a lipomatous hamartoma/hamartomatous lipoma, thought to be a suitable diagnosis of the lesion. Of most interest was that such a proliferating lesion occurred in the nasal turbinate.

Subconjunctival administration of bucillamine suppresses choroidal neovascularization in rat.

PURPOSE: Bucillamine is an antirheumatic drug with antiangiogenic properties that is currently used in clinical practice. Because bucillamine inhibits the production of VEGF, it is possible that this drug may inhibit choroidal neovascularization (CNV). Thus, the effect of bucillamine on the eyes of rats with experimental CNV was investigated in vivo by subconjunctival injection or oral intake. METHODS: CNV was induced in rat eyes by diode laser photocoagulation. The intensity of fluorescein leakage from the photocoagulated lesions was studied 7 and 14 days after photocoagulation. The areas of CNV lesions were measured histologically and studied immunohistochemically at days 4, 7, and 14. In addition, the concentration of the drug in ocular tissue and blood was measured by high-performance liquid chromatography-tandem mass spectrometry after the drug was delivered orally or subconjunctivally. RESULTS: After subconjunctival injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes throughout the study period. Histologic and immunohistochemical analyses 4, 7, and 14 days after photocoagulation demonstrated that the average size of the CNV lesions was reduced in the bucillamine-treated eyes compared with the control eyes. Subconjunctival injection maximized the ocular drug concentration while minimizing the blood concentration of the drug compared with oral intake. CONCLUSIONS: Subconjunctival injection of bucillamine significantly reduced the leakage and size of experimental CNV. These results suggest that bucillamine may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.