Effects of Anti-Glaucoma Prostaglandin Ophthalmic Solutions on Cultured Human Corneal Epithelial Cells.

Purpose: We compare the cytotoxicity of anti-glaucoma prostaglandin ophthalmic solutions on human corneal epithelial cells and elucidate mechanisms of toxicity. Methods: Cell viability was examined using MTS assay, and morphological changes of the cells were observed. Induction of necrosis/apoptosis was measured by colorimetric caspase assay. The production of Reactive oxygen species (ROS) and release of cytokines were analyzed using 2', 7'-dichlorodihydrofluorescein diacetate and bead-based indirect immunofluorescent assay, respectively. Results: Xalatan, Lumigan 0.01%, and Lumigan 0.03% decreased cell viability and induced morphological changes. Xalatan and Lumigan 0.01% induced necrosis. Xalatan, Lumigan 0.01%, Lumigan 0.03%, and Taflotan stimulated ROS production. Travatan and Lumigan 0.03% increased concentrations of Interleukin (IL)-6 and IL-8 in culture media. Conclusions: Xalatan and Lumigan 0.01% ophthalmic solutions demonstrated potent cytotoxicity compared with Lumigan 0.03%, Travatan, Taflotan, and Taflotan UD. Taflotan UD, compared to Taflotan 0.0015%, induced less oxidative stress and apoptotic signalling. The cytotoxicity might be partly associated with benzalkonium chloride.

Limited Azithromycin Localization to Rabbit Meibomian Glands Revealed by LC-MS-Based Bioanalysis and DESI Imaging.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye, and although it affects approximately 4% of the population, treatment options remain limited. Topical azithromycin is one of the most promising pharmacological agents because of its multiple mechanisms of action and long sustainability. Azithromycin is frequently used as an off-label medication in the U.S. However, although azithromycin is presumed to act directly on meibomian gland cells, the mechanisms of action that contribute to its clinical efficacy remain unclear because no studies using a pharmacokinetic approach have been performed. Therefore, we aimed to clarify whether topical azithromycin reaches the meibomian glands sufficiently to generate a biological effect. We measured azithromycin concentrations in rabbit meibomian glands collected using a recently developed method. Moreover, we also visualized the azithromycin micro-distribution using desorption electrospray ionization (DESI) imaging. Azithromycin concentration in the meibomian glands reached only 0.8 µg/g tissue following a single application of a 1% azithromycin ophthalmic solution and was 1000-fold lower than the concentration in conjunctival epithelium. Similarly, no signal was observed in the meibomian glands on DESI images. Our results clearly demonstrated that topical azithromycin had limited access to the meibomian glands and was predominantly distributed in ocular surface tissues such as the palpebral conjunctiva and lid margins. These findings provide new insight into the clinical responses to topical azithromycin therapy and will aid in the further development of effective drugs with more suitable pharmacokinetic properties.

Penetration Route of the Selective Glucocorticoid Receptor Agonist SA22465 and Betamethasone into Rabbit Meibomian Gland Based on Pharmacokinetics and Autoradiography.

Meibomian glands are modified sebaceous glands embedded within specific types of dense connective tissues. This study investigated drug penetration into meibomian glands following a single topical administration in rabbits. We measured time course concentrations of the selective glucocorticoid receptor agonist (SEGRA) SA22465 and betamethasone in lid margins, palpebral conjunctival epithelium, and meibomian glands following a single instillation using a newly established collection method. We also visualized the distribution of 14C-SA22465 in eyelid tissue sections using microautoradiography. Concentrations of SA22465 and its major metabolite SA22313 were highest in lid margins, followed by palpebral conjunctival epithelium and meibomian glands in a 100-fold descending order. Betamethasone exhibited similar distribution profiles with smaller concentration differences. The distribution of silver grains as a quantitative index of radioactivity in eyes and eyelids was determined in a subjective manner using microautoradiographs, which revealed that the highest distribution of silver grains was associated with the cornea, followed by posterior segment tissues, such as the sclera, choroid, and retina. Low levels were associated with more internal ocular tissues and a greater number of compartments. Moderate levels of radioactivity were associated with meibomian glands and connective tissues, including the nictitating membrane. In contrast, meibomian ducts contained only background levels of radioactivity. Our findings indicate that the transconjunctiva is the most likely route of drug entry into meibomian glands following ocular administration rather than the central meibomian duct; however, this distribution is limited. A physiologic barrier may restrict drug penetration across the tarsal plate.

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.

Functional Characterization of Carrier-Mediated Transport of Pravastatin across the Blood-Retinal Barrier in Rats.

Systemically administered pravastatin effectively treats diabetic retinopathy without central nervous system side effects. The efflux transport mechanism of pravastatin from the brain has already been clarified. In this study, the influx of pravastatin across the blood-retinal and blood-brain barriers (BRB and BBB) and the efflux of pravastatin from the retina were investigated using rats. Pravastatin influx (blood-to-tissues) was assessed using the retinal and brain uptake index (RUI and BUI) methods, and microdialysis was performed to investigate the efflux (retina-to-blood) transport of pravastatin. The RUI and BUI values for [(3)H]pravastatin were lower than those expected based on its lipophilicity, suggesting that the influx transport across the BRB and BBB was less than the reverse-direction transport. The RUI and BUI values for [(3)H]pravastatin were significantly decreased by pravastatin, digoxin, and probenecid, indicating that pravastatin undergoes carrier-mediated influx transport in the blood-to-tissues direction across the BRB and BBB. After intravitreal injection, [(3)H]pravastatin and the bulk flow marker [(14)C]d-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [(3)H]pravastatin during the terminal phase was 1.66-fold greater than that of [(14)C]d-mannitol. Efflux transport was reduced in the retinal presence of pravastatin, digoxin, and benzylpenicillin, suggesting that pravastatin is transported via efflux transporters. In conclusion, pravastatin is transported across the BRB via uptake and efflux transporters in both the blood-to-retina and retina-to-blood directions, and the retina-to-blood transporters are dominant, based on the lower values of the RUI compared with the values expected from the lipophilicity.

Benefits of Tafluprost and Timolol Fixed-Dose Combination for the Treatment of Glaucoma Are Confirmed by Studies on Experimental Animal Models.

PURPOSE: To assess the usefulness of 0.0015% tafluprost and 0.5% timolol fixed-dose combination (TT-FDC) for glaucoma, the ocular hypotensive effect of TT-FDC and concentration of tafluprost and timolol in the aqueous humor were compared with those of the concomitant administration of 0.0015% tafluprost and 0.5% timolol with or without an appropriate administration interval. METHODS: The ocular hypotensive effect was assessed by intraocular pressure (IOP) measurement in cynomolgus monkeys. Drug penetration into the aqueous humor was estimated by the concentrations of tafluprost acid (active metabolic form of tafluprost) and timolol, which were measured using liquid chromatography-tandem mass spectrometry after administration of tafluprost and timolol to Sprague Dawley rats. RESULTS: The ocular hypotensive effect of TT-FDC was equivalent to that of the concomitant administration of timolol and tafluprost at a more than 5-min interval in monkeys. However, the ocular hypotensive effect of the concomitant administration of timolol and tafluprost without an interval (-2.8 ± 0.2 mmHg at peak IOP reduction) was significantly weaker compared with TT-FDC (-4.3 ± 0.5 mmHg at peak IOP reduction, P = 0.008 vs. concomitant administration of timolol and tafluprost) in monkeys. The aqueous humor concentration of the second administered drug (tafluprost) was not affected by the dosing conditions, whereas the concentration of the first instilled drug (timolol) without the interval was lower than that with a 5-min interval (1,200 ng · h/mL vs. 1,890 ng · h/mL in AUC0-4) in rats. CONCLUSION: TT-FDC demonstrates a clear benefit by preventing efficacy loss without an appropriate interval in experimental animal models.

Expression of the P2Y2 receptor on the rat ocular surface during a 1-year rearing period.

PURPOSE: P2Y2 receptors are expressed on ocular surface tissues. Diquafosol ophthalmic solution (DIQUAS(®) ophthalmic solution 3 %; Santen Pharmaceutical Co., Ltd.) acts on these receptors and promotes the secretion of water and mucin. It has been shown to be an efficient dry eye treatment. If P2Y2 receptor expression on the ocular surface decreases with age, the effect of diquafosol may be reduced in elderly persons. In this study, we investigated the changes in P2Y2 receptor expression on the rat ocular surface over an extended period of time. METHODS: P2Y2 receptor expression in the conjunctiva, cornea, meibomian gland and lacrimal glands of male and female Sprague-Dawley rats was examined from 5 weeks until 53 weeks of age using immunostaining and quantitative-PCR. RESULTS: In the immunohistological examinations, P2Y2 receptor expression was observed in the conjunctival epithelium containing goblet cells, corneal epithelium, meibomian gland ductal epithelium and lacrimal gland ductal epithelium. However, its expression was not significantly different between each age group or between sexes. Regarding P2Y2 receptor mRNA expression, there was an age-related increase in the bulbar conjunctiva. In particular, a significant increase was observed in the 53-week-old age group as compared to the 5-week-old female age group. However, age-related changes in expression were not observed in the cornea or meibomian gland in males or females. CONCLUSIONS: We observed no significant age-related decrease was observed for P2Y2 receptor protein and mRNA expression on rat ocular surface tissues.

Impact of P-glycoprotein on blood-retinal barrier permeability: comparison of blood-aqueous humor and blood-brain barrier using mdr1a knockout rats.

PURPOSE: The purpose of this study was to clarify the impact of P-glycoprotein (P-gp) on blood-retinal barrier (BRB) and blood-aqueous humor barrier (BAB) permeability, in contrast to blood-brain barrier (BBB) permeability. METHODS: Permeabilities of six compounds, including P-gp substrates (quinidine, digoxin, and verapamil), were investigated in wild-type and mdr1a knockout rats using retinal, aqueous humor, and brain uptake index (RUI, AHUI, and BUI, respectively) methods and integration plot analysis. RESULTS: In both rat strains, quinidine, digoxin, and verapamil were transported by P-gp across each barrier; however, the impact of P-gp on retinal uptake of quinidine and verapamil was less pronounced than that on brain uptake. The apparent influx permeability clearance (Kin) values of verapamil in retina obtained from wild-type and knockout rats were similar (0.824 ± 0.201 and 0.849 ± 0.980 mL/min·g retina, respectively; mean ± SD; n = 3 rats). The Kin in aqueous humor and brain obtained from knockout rats was, respectively, 3-fold and 12-fold higher than that of wild-type (P < 0.05). In P-gp-deficient conditions, the RUI and AHUI of quinidine, digoxin, and verapamil, as well as the BUI of quinidine and digoxin, were decreased by P-gp inhibitors. However, the BUI of verapamil was not changed by P-gp inhibitors. Results suggest that carrier-mediated influx transporters exist in the blood-ocular barriers and that the function of verapamil influx transporters is markedly different between the retina and brain. CONCLUSIONS: In both rat strains, P-gp operates in the blood-ocular barriers, and the impact of P-gp on BRB permeability to quinidine and verapamil is lower than that on BBB permeability.

Rapid identification of fatty acids and (O-acyl)-ω-hydroxy fatty acids in human meibum by liquid chromatography/high-resolution mass spectrometry.

We report a rapid liquid chromatography/quadrupole Orbitrap Fourier transform mass spectrometry (LC-FTMS) method for identifying free fatty acids (FAs) and (O-acyl)-ω-hydroxyFAs (OAHFAs) in human meibum without derivatization. Meibum is a lipid-rich secretion and an important component of the tear film lipid layer. FAs are commonly detected by gas chromatography (GC) or GC/MS after methyl ester derivatization. We developed high-throughput lipid profiling using LC-FTMS and lipid identification software, Lipid Search, without derivatization and applied the method to human meibum. Chromatographic separation was performed on a C18 column. We selected negative electrospray ionization [M-H](-), and applied high-resolution full scan mode to FA analysis and data-dependent MS(2) mode to OAHFA analysis. High-resolution Orbitrap MS proved to be an excellent tool for the rapid analysis of lipids from meibum, and 100 FA and 61 OAHFA molecular species were detected. The analysis times were 12 and 16.5min, respectively. Very long chain FAs (up to C37) and OAHFAs (up to C56) were also detected. The results clearly showed that retention time correlates with the number of double bonds and carbon chains. This LC/MS method can be applied to the identification of FAs and OAHFAs in human meibum.

Local toxicity of benzalkonium chloride in ophthalmic solutions following repeated applications.

We performed repeated toxicity studies of benzalkonium chloride (BAK)-containing vehicles of ophthalmic solutions in monkeys and rabbits to assess the local toxicity of BAK after repeated applications on the ocular surface. Local toxicity of BAK was evaluated by toxicity studies in which a 0.01% BAK-containing vehicle was applied twice/day for 52 weeks, 4 times/day for 39 weeks, or 6 times/day for 13 weeks, or in which a 0.005% BAK-containing vehicle was applied 6 times/day for 52 weeks or twice/day for 4 weeks in monkeys. Local toxicity of BAK was also evaluated where a 0.01% BAK-containing vehicle was applied 6 times/day for 6 weeks, or a 0.005% BAK-containing vehicle was applied twice/day for 39 weeks or 8 times/day for 4 weeks in rabbits. These doses were chosen because BAK is generally used at concentrations up to 0.01% in ophthalmic solutions. The BAK-containing vehicle did not cause ophthalmological changes suggestive of irritation, allergy, or corneal damage. We also did not observe any histopathological changes in the eyeball, eyelid, lacrimal gland, and nasal cavity, with repeated applications of BAK for up to 52 weeks, up to 8 times/day, or at concentrations up to 0.01%, in monkeys and rabbits. Our results suggest that BAK in concentrations up to 0.01% in ophthalmic solution is non-toxic to the eyeball, its accessory organs, and the nasal cavity after long repeated applications.

Characterization of monocarboxylate uptake and immunohistochemical demonstration of monocarboxylate transporters in cultured rabbit corneal epithelial cells.

OBJECTIVES: This study aimed to characterize the mechanisms of monocarboxylate uptake by cultured rabbit corneal epithelial cells (RCECs) using l- and d-lactic acids as model substrates. METHODS: l-/d-Lactic acid uptake was evaluated by measuring the accumulation in confluent RCECs. Also, we demonstrated the distribution of monocarboxylate transporters (MCTs) in RCECs by immunohistochemistry. KEY FINDINGS: The accumulation of (14) C-labelled l- and d-lactic acids was dependent on time, pH and temperature. The Arrhenius plots of the uptake were biphasic. The initial uptake of (14) C-labelled l-lactic acid exhibited concentration dependence and was greater than that of the d-isomer. The initial uptake of (14) C-labelled l- and d-lactic acids involved saturable and nonsaturable processes; the saturable process exhibited higher affinity for l-lactic acid than for the d-isomer. l-/d-lactic acid uptake was inhibited by chiral monocarboxylate in a stereoselective manner. The uptake of (14) C-labelled l- and d-lactic acids was sensitive to metabolic inhibitors and other monocarboxylates. MCT expression in RCECs was confirmed immunohistochemically. In particular, MCT2 expression was detected in RCECs, whereas MCT1, MCT4 and MCT5 expression was detected in the surface layer. CONCLUSION: These results indicate that the carrier-mediated transport system specific for monocarboxylates elicits lactic acid uptake in RCECs. Therefore, the transcorneal permeation of drugs with a monocarboxylic moiety may be dependent on the activity of a specific pH-dependent transporter as well as passive diffusion according to the pH-partition theory.

Characterization of the carrier-mediated transport of ketoprofen, a nonsteroidal anti-inflammatory drug, in rabbit corneal epithelium cells.

OBJECTIVES: Using rabbit corneal epithelial cells (RCECs), the transport of a nonsteroidal anti-inflammatory drug (NSAID) [(3)H]ketoprofen across the cornea was investigated with the aim of revealing the mechanism of uptake. METHODS: [(3)H]Ketoprofen transport was evaluated by measuring the permeability across the RCECs layers. KEY FINDINGS: [(3)H]Ketoprofen uptake was time, temperature and pH dependent. Maximal uptake occurred from a solution with a pH of 5.25. Uptake was also reduced by metabolic inhibitors (sodium azide and dinitrophenol (DNP)) and proton-linked monocarboxylate transporter (MCT) inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and α-cyano-4-hydroxycinnamic acid (CHC)). [(3)H]Ketoprofen uptake was significantly inhibited by various monocarboxylates and other NSAIDs and by MCT and/or organic anion transporter (OAT) inhibitors probenecid and p-aminohippurate, but was unaffected by organic anion-transporting polypeptide (OATP) inhibitors bromosulfophthalein and taurocholate. The specific uptake of [(3)H]ketoprofen was saturable. Eadie-Hofstee plots indicated the involvement of high- and low-affinity components. The K(m) and V(max) values for the high- and low-affinity components of [(3)H]ketoprofen uptake were 0.56 and 24 mm, and 0.37 and 61 nmol/min/mg of protein, respectively. Benzoic acid, a substrate and inhibitor of MCTs, selectively inhibited low-affinity [(3)H]ketoprofen uptake. Conversely, indometacin inhibited high-affinity [(3)H]ketoprofen uptake. CONCLUSION: The results of this study suggest that the monocarboxylate transport system partly accounts for the low-affinity component of [(3)H]ketoprofen uptake, and that the carrier-mediated transport systems such as the OAT family, shared by NSAIDs account for the high-affinity component.

Ocular toxicity of benzalkonium chloride homologs compared with their mixtures.

This study was performed to assess the in vivo ocular toxicity of benzalkonium chloride (BAK) homologs compared with commercially available BAK (BAK mixture) and to assess the ocular toxicity of BAK homolog after repeated ocular application. Rabbit eyes were examined by ophthalmology and scanning electron microscopy (SEM) after 10 applications of BAK homologs with C12 (C12-BAK) and C14 (C14-BAK) alkyl chain lengths and a BAK mixture at concentrations of 0.001% (w/v), 0.003% (w/v), 0.005% (w/v), 0.01% (w/v) and 0.03% (w/v). The ocular toxicity of C12-BAK to rabbit eyes was examined by ophthalmology and histopathology after repeated ocular application for 39 weeks. In addition, the antimicrobial activities of C12-BAK and C14-BAK against A. niger, S. aureus and P. aeruginosa were assessed. Ocular toxicity of C12-BAK was less than those of the BAK mixture and C14-BAK. No ocular toxicity was noted after ocular application of 0.01% C12-BAK to rabbits for 39 weeks. C12-BAK showed antimicrobial activities at a concentration of 0.003%. These results suggest that the use of C12-BAK to replace BAK mixture as a preservative in ophthalmic solutions should be considered in order to reduce the incidence of the corneal epithelial cell injury induced clinically by BAK.

Tolerability and pharmacokinetics of intravitreal sirolimus.

PURPOSE: To evaluate the pharmacokinetics (PK) and tolerability of a proprietary sirolimus depot-forming ocular formulation in rabbits and humans after a single intravitreal (i.v.t.) injection. METHODS: New Zealand White (NZW) rabbits were intravitreally injected in both eyes with an injectable formulation in 5 (3 PK and 2 tolerability) studies. The rabbits received up to approximately 220 µg sirolimus per eye. At the desired timing post-injection, the animals were euthanized; both eyes were enucleated, frozen, and dissected to separate sclera, retina/choroid, and vitreous humor (VH). Whole blood (WB) samples were obtained at each time point before euthanasia. In clinical trials, patients received an i.v.t. injection of approximately 352 µg sirolimus. Sirolimus concentrations in ocular tissues and WB samples were measured using liquid chromatography/tandem mass spectrometry (LC/MS/MS). In both single- and repeat-dose tolerability studies, systemic and ocular adverse effects were evaluated. RESULTS: After i.v.t. administration, sirolimus formed a depot in the VH. During dissolution, concentrations in VH were dose related and exhibited continuous release from the depot. This was characterized by a gradient of sirolimus concentration in the order of VH > retina/choroid > sclera > WB, and the concentrations were maintained for approximately 2 months after the i.v.t. injection. After repeat dosing (132 µg), no drug accumulation was seen in the ocular tissue or systemically. In clinical studies, the highest blood levels were <2 ng/mL at day 2, and half-time (t(1/2)) was 8-9 days. There was no accumulation at day 30 after the i.v.t. injection (up to 352 µg). Safety studies conducted on rabbits indicated good local tolerability. Sirolimus-related effects were limited to minor incipient cataract findings and mild lenticular changes. In the clinical studies where sirolimus was intravitreally administered up to 352 µg, injections were well tolerated. CONCLUSIONS: Sustained i.v.t. delivery was achieved in a dose-dependent fashion after the i.v.t. injection of a proprietary sirolimus depot-forming ocular formulation. Across the tolerability and safety studies, no significant findings were observed for systemic and ocular tolerability. The human WB levels were well below the daily trough systemic blood level range required for systemic immunosuppression. An i.v.t. injection of sirolimus has a PK and safety profile that is favorable for treating inflammatory conditions of the eye, such as non-infectious uveitis, and warrants further investigation in humans.

Comparison of drug permeabilities across the blood-retinal barrier, blood-aqueous humor barrier, and blood-brain barrier.

Drugs vary in their ability to permeate the blood-retinal barrier (BRB), blood-aqueous humor barrier (BAB), and blood-brain barrier (BBB) and the factors affecting the drug permeation remain unclear. In this study, the permeability of various substances across BRB, BAB, and BBB in rats was determined using the brain uptake index (BUI), retinal uptake index (RUI), and aqueous humor uptake index (AHUI) methods. Lipophilic substances showed high permeabilities across BBB and BRB. The RUI values of these substances were approximately four-fold higher than the BUI values. The AHUI versus lipophilicity curve had a parabolic shape with AHUI(max) values at log D(7.4) ranging from -1.0 to 0.0. On the basis of the difference on the lipophilicities, verapamil, quinidine, and digoxin showed lower permeability than predicted from those across BBB and BRB, whereas only digoxin showed a lower permeability across BRB. These low permeabilities were significantly increased by P-glycoprotein inhibitors. Furthermore, anion transporter inhibition increased the absorption of digoxin to permeate into the retina and aqueous humor. In conclusion, this study suggests that efflux transport systems play an important role in the ocular absorption of drugs from the circulating blood after systemic administration.

Metabolism and ocular tissue distribution of an antiglaucoma prostanoid, tafluprost, after ocular instillation to monkeys.

PURPOSE: To investigate the metabolism of a new antiglaucoma difluoroprostaglandin, tafluprost, in ocular tissues and evaluate the distribution of the parent drug and its metabolites in ocular and systemic tissues after a single ocular administration to cynomolgus monkeys (Macaca fascicularis). METHODS: A single dose of an ophthalmic solution containing 0.0005%, 0.005%, or 0.05% [(3)H]tafluprost was topically instilled (20 µL/eye) to male and/or female cynomolgus monkeys to study tissue distribution and metabolism. Blood, ocular/systemic tissues, or excreta were collected until 24 h after dosing. The radioactivity of each sample was measured by liquid scintillation counting, and metabolites were characterized by liquid chromatography-mass spectrometry. The major metabolites found in ocular tissues were intracameraly administered to monkeys to confirm their effect on intraocular pressure (IOP). RESULTS: Soon after dosing, high concentrations of drug-related radioactivity were observed in the cornea and bulbar/palpebral conjunctiva, followed by the iris, sclera, choroid with retinal pigmented epithelium, and aqueous humor. The highest concentration of radioactivity concentrations occurred in the anterior and posterior ocular tissues within 2 h after dosing. The radioactivity measured in the plasma and ocular tissues was proportional to the dose administered. The major metabolites of tafluprost identified in the ocular tissues were tafluprost acid and 1,2-dinor- and 1,2,3,4-tetaranor-tafluprost acid. The estimated concentration of tafluprost acid in the aqueous humor and ciliary body was enough to stimulate prostanoid FP-receptors. After hydrolysis to the acid form, the primary metabolic pathway of tafluprost was via ß-oxidation and, subsequently, oxidation. No metabolic reactions to the 15-carbon position were observed. Tafluprost acid was shown to significantly lower the IOP, whereas 1,2-dinor- and 1,2,3,4-tetaranor-tafluprost acid did not. CONCLUSIONS: Topically administered [(3)H]tafluprost was well absorbed into the ocular and systemic tissues of the primary nonclinical species, monkey. The amount of the pharmacologically active form, that is, tafluprost acid, was high enough to occupy the target FP receptors at the site of action. The pharmacokinetic and metabolic properties of this difluorinated prostaglandin in primates are believed to result in clinical benefits of a long-term IOP-lowering effect.

Ocular pharmacokinetic/pharmacodynamic modeling for multiple anti-glaucoma drugs.

We have constructed a new ocular pharmacokinetic pharmacodynamic (PK/PD) model for anti-glaucoma drugs to describe ocular hypotensive effects on intraocular pressure (IOP) after instillation of a combination of an alpha(1)-adrenergic antagonist, bunazosin, and a beta-adrenergic antagonist, timolol, into rabbits. This model was constructed by the combination of two ocular PK/PD models for bunazosin and timolol by including aqueous humor dynamics based on both action mechanisms. We also verified the reliability of this model by confirming the drug concentrations in aqueous humor and ocular hypotensive effects after instillation of the drug combination. The aqueous humor concentrations of timolol and bunazosin were determined by an HPLC, and ocular hypotensive effect-time profiles were measured using a telemetry system, which was able to record automatically detailed effects. The combined model could simulate the aqueous humor concentrations of both drugs and the additive IOP-lowering effect after instillation of the combination using the MULTI (RUNGE) program and PK/PD parameters which were obtained from ocular hypotensive effects after instillation of bunazosin alone or timolol alone. The theoretical concentration curves of both drugs in the aqueous humor and the theoretical ocular hypotensive effect curves almost agreed with both the observed concentrations and ocular hypotensive effects after instillation of the drug combination. These results indicate the reliability and usefulness of PK/PD modeling considering aqueous humor dynamics to predict IOP in multidrug therapy. This is the first study to develop a PK/PD model for multidrug therapy for the eye.

Ocular pharmacokinetic/pharmacodynamic modeling for timolol in rabbits using a telemetry system.

We have established an ocular pharmacokinetic/pharmacodynamic (PK/PD) model for a beta-adrenergic antagonist, timolol, after instillation into rabbits. Timolol concentrations were determined by HPLC in the tear fluid, aqueous humor, cornea, and iris-ciliary body after instillation or ocular injection into the anterior chamber of the eye in rabbits. In addition, intraocular pressure (IOP) measurement was performed after instillation of timolol by a telemetry system, which was able to obtain detailed IOP data automatically. The PK/PD parameters were estimated by fitting the concentration-time profiles and the ocular hypotensive effect-time profiles using MULTI (RUNGE) program. The PK model consisted of six compartments and the PD model included aqueous humor dynamics based on an action mechanism of timolol, which causes lowering of IOP by suppressing aqueous humor production. The PK/PD model described well the concentration-time profiles and the ocular hypotensive effect-time profiles after instillation of timolol. This study is the first trial to develop an ocular PK/PD model for timolol after instillation. This model can predict both the drug concentrations in various ocular tissues and the ocular hypotensive effect after instillation of timolol.

Transport of timolol and tilisolol in rabbit corneal epithelium.

The purpose of this study is to characterize the transport of tilisolol and timolol through the corneal epithelium, which is believed to be a tight barrier of ocular drug absorption. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Beta-blocker permeability through the RCEC layer was measured to assess the transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. The transcellular permeability of tilisolol was dependent on drug concentration although timolol showed no concentration dependency. Tilisolol flux from the apical to the basal side was larger than in the opposite direction although timolol showed no direction dependency. The transcellular permeability of tilisolol from the apical to the basal side was inhibited by sodium azide, tetraethylammonium, quinidine, taurocholic acid, guanidine and carnitine. Tilisolol had an active mechanism in uptake to the corneal epithelium, probably by the organic cation transporter family, although timolol predominantly permeated via passive diffusion. This RCEC system was useful to characterize the ocular permeation mechanism of drugs.

Transport of acebutolol through rabbit corneal epithelium.

The purpose of this study is to characterize transport of acebutolol through the corneal epithelium. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate the drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Measurements of acebutolol permeability through RCEC layer were carried out to assess transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. Paracellular permeability coefficient (P(paracell)) was calculated by permeability coefficient of hydrophilic drugs (P(cell)). The transcellular permeability of acebutolol from apical side to basal side (A-to-B) showed concentration-dependency. The acebutolol flux in the A-to-B direction was smaller than that of opposite direction. Sodium azide, verapamil, and cyclosporin A enhanced the transcellular permeability of acebutolol in the A-to-B direction. Acebutolol permeability through an excised rabbit cornea was also increased by verapamil. Thus, it was suggested that acebutolol was actively secreted via P-glycoprotein in a corneal epithelium.