Kouichi Kawazu

Protective effects of timolol against the neuronal damage induced by glutamate and ischemia in the rat retina

The purpose of this study was to determine whether timolol, an ocular hypotensive drug, has retinal neuroprotective effects in experimental in vitro and in vivo models. For in vitro studies, we used retinal neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the former, neurotoxicity was induced using 1 mM glutamate and cell viability was assessed. In RGCs, neurotoxicity was induced using 25 microM glutamate for 3 days and cell viability was assessed. For the in vivo study, we used a rat model of retinal ischemic injury induced by elevating intraocular pressure (IOP) by raising the hydrostatic pressure. The retinal damage was evaluated by counting the number of cells in the ganglion cell layer (GCL) and by examining the a- and b-waves in the electroretinogram (ERG). For the intraocular distribution study, 0.5% [3H]timolol was topically applied to rat eyes, and these were enucleated after various intervals and divided into parts. Each part was combusted and the radioactivity measured. Timolol (0.1 and 1 microM) markedly reduced the glutamate-induced neuronal cells in retinal neuron cultures and in RGCs. After ischemic-reperfusion, both the number of cells in the GCL and a- and b-waves in the ERG decreased; however, topically applied 0.5% timolol reduced these effects. Topically applied 0.5% timolol was detected at a concentration of approximately 1 microg/g wet tissue in retina-choroid at 30 min after its application. In conclusion, timolol was effective against retinal neuron damage both in vitro and in vivo. Furthermore, topically applied timolol reached the retina-choroid. These findings suggest that timolol has a direct neuroprotective effect against retinal damage.

Characterization of the carrier-mediated transport of levofloxacin, a fluoroquinolone antimicrobial agent, in rabbit cornea.

The cornea presents a formidable barrier to drug penetration. The fluoroquinolone levofloxacin, which is an effective antimicrobial agent, has the potential to be used in the topical treatment of ocular disease. Thus, we sought to characterize how levofloxacin penetrates the cornea. To perform this characterization, we measured the time dependent permeation of levofloxacin across the isolated rabbit cornea using a diffusion chamber, and compared it with antipyrine fluxes.

Disposition and Metabolism of a Novel Prostanoid Antiglaucoma Medication, Tafluprost, Following Ocular Administration to Rats

The disposition and metabolism of tafluprost, an ester prodrug of the 15,15-difluoro-prostaglandin F2α antiglaucoma agent, have been studied in rats after ocular administration. Radioactivity was absorbed very rapidly into the eye and systemic circulation after a single ocular dose of 0.005% [3H]tafluprost ophthalmic solution, with maximum levels in plasma and most eye tissues occurring within 15 min. The absorption ratio of radioactivity was approximately 75%, suggesting the high availability of ocular administration of tafluprost. Approximately 10% of the dose was present in cornea at this time, and radioactivity concentrations in this tissue exceeded those in aqueous humor and iris/ciliary body throughout the 24-h study period. After repeated daily ocular doses, radioactivity levels remained greatest in cornea, followed by iris/ciliary body that replaced aqueous humor as the eye tissue containing the second highest radioactivity concentration. In female rats, radioactivity was excreted equally between urine and feces after a single ocular dose, whereas in male rats more was excreted in feces, reflecting the greater biliary excretion in males rats (50% dose) compared with females rats (33% dose). Tafluprost was extensively metabolized in the rat, such that intact prodrug was not detected in plasma, tissues, or excreta by radio-high-performance liquid chromatography. On the other hand, the active moiety, tafluprost acid, was the only noteworthy radioactive component in cornea, aqueous humor, and iris/ciliary body for at least 8 h after the ocular dose, and it was also a major plasma metabolite in early time points. The gender differences in conjugation reactions resulted in the differences in the excretion.

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.

Cultured rabbit corneal epithelium elicits levofloxacin absorption and secretion.

Evidence for carrier‐mediated transport of levofloxacin in the isolated rabbit cornea has been found. However, it is not known whether this mechanism is located in the epithelium or the endothelium. To resolve this question, we have measured the kinetics of levofloxacin uptake in primary cultures of rabbit corneal epithelial cells.

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.

Ocular pharmacokinetic/ pharmacodynamic modeling for bunazosin after instillation into rabbits.

AbstractPurpose. To develop a pharmacokinetic/pharmacodynamic (PK/PD) model for an α1-blocker (bunazosin) after instillation. The PK/PD model can predict both the drug concentrations in various ocular tissues and the hypotensive effect. Methods. Bunazosin concentrations were determined with High Performance Liquid Chromatography (HPLC) in tear fluid, the aqueous humor, cornea, and iris-ciliary body after instillation or ocular injection into the anterior chamber in rabbits. After instillation of bunazosin in rabbits, intraocular pressure (IOP) was also determined with a pneumatic tonometer. The PK/PD parameters were estimated by fitting the concentration-time profiles and the hypotensive effect-time profiles to the developed PK/PD models using the MULTI (RUNGE) program. Results. On the basis of the concentration-time profiles of bunazosin, a PK model, including seven compartments, was developed for examining the behavior of bunazosin after instillation. Then, two PK/PD models for hypotensive effect of bunazosin were developed using an indirect response (model A) and the relationship between IOP and aqueous humor flow (model B). These models well described the concentration-time profiles and hypotensive effect-time profiles of bunazosin after instillation. Conclusions. This study is the first trial to develop a PK/PD model for an antiglaucoma agent using an indirect response and the relationship between IOP and aqueous humor flow.

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 β-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.

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 [3H]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 [3H]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, [3H]pravastatin and the bulk flow marker [14C]d-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [3H]pravastatin during the terminal phase was 1.66-fold greater than that of [14C]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

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

Using rabbit corneal epithelial cells (RCECs), the transport of a nonsteroidal anti‐inflammatory drug (NSAID) [3H]ketoprofen across the cornea was investigated with the aim of revealing the mechanism of uptake.