Hideki Tokushige

Intraocular pressure-lowering effects and safety of topical administration of a selective ROCK inhibitor, SNJ-1656, in healthy volunteers.

OBJECTIVE To investigate the effects and safety of topical administration of an ophthalmic solution of a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor, SNJ-1656, 0.003% to 0.1%, in healthy male adult volunteers. DESIGN Randomized, double-masked, group-comparison, phase 1 clinical study. In the initial single-instillation trial, 45 healthy volunteers were randomly subdivided into 5 groups and treated with SNJ-1656 in concentrations of 0.003%, 0.01%, 0.03%, 0.05%, and 0.1% in stepwise fashion. In the repeated-instillation trial, 36 healthy volunteers were assigned to receive SNJ-1656 ophthalmic solution at the following concentrations and dosages: 0.05% once daily, 0.1% once daily, 0.05% twice daily, or 0.1% twice daily. In our studies, the administration of the solution and subsequent examinations (including intraocular pressure [IOP] measurements) were performed in a double-masked fashion. RESULTS After single instillation of placebo or SNJ-1656, in concentrations of 0.003%, 0.01%, 0.03%, 0.05%, and 0.1%, the changes in IOP from the baseline were -0.91, -1.18, -1.48, - 2.20 (P = .04 vs placebo), -1.48, and -1.98 mm Hg, respectively, at 2 hours, and -0.63,-0.95, -1.79, -2.26 (P = .01 vs placebo), -1.95, and -3.00 mm Hg (P < .001 vs placebo) respectively, at 4 hours. Significant IOP reductions after repeated instillation were also found. On slitlamp examination during the trial, there were no significant adverse findings except hyperemia of the bulbar and palpebral conjunctiva after instillation. CONCLUSION This clinical study demonstrated that SNJ-1656 is a safe topical agent effective in reducing IOP in human eyes.

Effects of Y-39983, a Selective Rho-Associated Protein Kinase Inhibitor, on Blood Flow in Optic Nerve Head in Rabbits and Axonal Regeneration of Retinal Ganglion Cells in Rats

Purpose: To investigate the effects of Y-39983, a selective Rho-associated coiled coil-forming protein kinase inhibitor, on blood flow in the optic nerve head (ONH) in rabbits and axonal regeneration of retinal ganglion cells (RGCs) in rats. Methods: Blood flow in ONH was measured by the laser speckle method after topical administration of 0.05% Y-39983 solution or its vehicle in rabbit eyes. To investigate the effects of Y-39983 on axonal regeneration of RGCs, RGCs purified from rat eyes were cultured with or without 10 μM Y-39983 and morphologically observed by phase-contrast microscopy. Moreover, the effects of intravitreal administration of Y-39983 were evaluated using an in vivo model of axotomized RGCs in peripheral nerve-grafted rats. Results: Topical administration of 0.05% Y-39983 solution significantly increased blood flow in ONH compared with the vehicle group in rabbits. Maximum increase in blood flow in the 0.05% Y-39983 group was 122.84 ± 5.98 % (Mean ± S.E.) at 90 minutes after administration compared with before administration. Neurites in rat RGCs treated with 10 μM Y-39983 were extended compared with those without Y-39983 treatment of RGCs in vitro. Y-39983 dose-dependently increased the number of RGCs with regenerating axons in vivo. The numbers of RGCs with regenerating axons in 10 and 100 μM Y-39983-treated rats were 99.3 ± 10.5 and 169.5 ± 43.3 cells/mm2 (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm2). Conclusion: Y-39983 may be a candidate drug not only for lowering of IOP but also for increasing of blood flow in ONH in the treatment of glaucoma. Moreover, Y-39983 may have therapeutic potential for axonal regeneration of RGCs in the treatment of diseases with degenerating axons of RGCs including glaucoma, although improvements of formulation or route of administration are needed in order to reach an effective concentration in retina.

Immunomodulatory Effect of Gatifloxacin on Mouse Peritoneal Macrophages in vitro and in Models of Endotoxin-Induced Rat Conjunctivitis and Rabbit Bacterial Keratitis

Aim: To determine the anti-inflammatory activity of gatifloxacin in ophthalmic use. Methods: The following 3 experiments were carried out. (1) Rabbits were inoculated intracorneally with methicillin-resistant Staphylococcus aureus and topically treated with gatifloxacin or levofloxacin. The severity of infection and viable bacterial count were assessed. (2) Thioglycollate-elicited mouse peritoneal macrophages were stimulated by Pseudomonas lipopolysaccharides (LPS) in the presence of graded concentrations of fluoroquinolones, and macrophage-derived tumor necrosis factor α (TNF-α) was assessed. (3) The effects of fluoroquinolones on TNF-α production were compared in an LPS-induced rat conjunctivitis model. Results: In the rabbit keratitis model, the ocular inflammation was significantly reduced by gatifloxacin as compared to levofloxacin but there was no significant difference between the groups in the number of viable bacteria. Gatifloxacin and levofloxacin suppressed TNF-α production in mouse macrophages in a concentration-dependent manner, and the effect of gatifloxacin was more potent than that of levofloxacin. Moxifloxacin exhibited no effect in this condition. In the rat conjunctivitis model, the tissue TNF-α level was significantly reduced only in the group instilled with gatifloxacin ophthalmic solution. Conclusion: These results indicate that gatifloxacin has not only antibacterial activity but an anti-inflammatory action caused by at least inhibiting TNF-α production at the doses used in topical ophthalmic therapy.

Efficacy and safety of SNJ-1656 in primary open-angle glaucoma or ocular hypertension.

first decade of life. A further recent paper detailed a consanguinous family with early onset cone-rod dystrophy due to splice site mutations in ADAM9 (El-Haig et al. 2014). We report the detailed clinical phenotype in a child with retinal disease caused by a homozygous mutation in ADAM9. A male patient presented at age 3 years. He was noted in infancy to have a right convergent squint with poor vision and eccentric fixation. There was no nystagmus. He was otherwise well with normal development. The parents were from Pakistan and were first cousins. At last review, age 7, the vision was right 1.0 logMAR (Snellen 6/60), left 0.88 logMAR (Snellen 6/48) with a hyperopic, astigmatic refractive error of R + 4.00/ 2.50 9 20 L + 4.00/ 2.00 9 180. There was a moderate left divergent squint with eccentric fixation. Early posterior subcapsular cataract was noted. Fundus examination showed posterior pole atrophy with a white-speckled appearance, which extended to the arcades and encompassed the optic disc (Fig. 1). Retinal imaging demonstrated reduced autofluorescence in the posterior pole with atrophy of the outer retina on optical coherence tomography (Fig. 1). Pattern and flash electroretinography (PERG; ERG) performed at the age of 3 years using surface electrodes revealed an undetectable PERG and borderline photopic and scotopic ERGs. At the age of 7 years, the PERG and full-field ERG were performed using corneal electrodes to ISCEV standards. The PERG was undetectable in keeping with severe macular dysfunction, and mildly abnormal full-field ERGs were consistent with cone-rod dystrophy (Fig. 1). Whole exome sequencing was performed (AROS Applied Biotechnology, Aarhus, Denmark), which identified a novel, homozygous mutation in ADAM9, c.967delT; p.Ser323Glnfs*33, which on direct Sanger sequencing was shown to segregate in the family with both parents heterozygous for this mutation (Fig. 1). Non-syndromic, autosomal recessive CORD is rare and usually associated with biallelic mutations in ABCA4 (Bocquet et al. 2013). In the previous reports of CORD due to ADAM9, five families were identified with mutations leading to either aberrant splicing or premature truncation codons. Similar to our patient, all had poor vision in their first decade of life, no nystagmus and outer retinal atrophy of the macula. Most were also noted to have discrete white patches in the posterior pole and around the disc and a peripheral pigmentary retinopathy, which is not present in our patient. Retinal imaging in a previous report of the index family demonstrated posterior pole atrophy in two patients in their 40s with a similar appearance to our patient (Danciger et al. 2001). Electrophysiology in these two patients demonstrated severe loss of both cone and rod function. In the recent report of a single family, the youngest patient assessed was 17 years (El-Haig et al. 2014). Posterior pole atrophy was noted, and in the retinal images this also encompassed the disc. No electrophysiology was available. Given the young age of our patient, we have been able to demonstrate the electrophysiological phenotype of severe loss of macular function in the early stages with relatively mild peripheral retinal dysfunction. In both a canine and mouse model, there are cone and rod photoreceptor abnormalities on electrophysiology which are not apparent in very young animals but develop with time (Parry et al. 2009; Goldstein et al. 2010). In both models, histopathology localized the primary defect to the apical microvilli of the retinal pigment epithelium potentially mediated by failure of normal photoreceptor outer segment phagocytosis. These animal models show early preservation of photoreceptor structure despite dysfunction. This together with relatively good peripheral photoreceptor function in early human disease as illustrated by this case suggests that there is a therapeutic window for gene therapy in patients with mutations in ADAM9.

Pharmacokinetics and efficacy of topically applied nonsteroidal anti-inflammatory drugs in retinochoroidal tissues in rabbits.

Purpose To evaluate the pharmacokinetics and efficacy of topically applied nonsteroidal anti-inflammatory drugs (NSAIDs) in the retinochoroidal tissues of rabbits. Methods The cyclooxygenase (COX) inhibitory activity of diclofenac, bromfenac, and amfenac, an active metabolite of nepafenac, were determined using human-derived COX-1 and COX-2. Each of the three NSAIDs was applied topically to rabbits, and after 0.5 to 8 hrs, the concentration of each drug in the aqueous humor and the retinochoroidal tissues was measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetics of the drugs in the tissues after repeated doses as is done on patients was calculated by a simulation software. The inhibitory effect of each NSAID on the breakdown of the blood-retinal barrier was assessed by the vitreous protein concentration on concanavalin A-induced retinochoroidal inflammation in rabbits. Results The half-maximal inhibitory concentration (IC50) of diclofenac, bromfenac, and amfenac was 55.5, 5.56, and 15.3 nM for human COX-1, and 30.7, 7.45, and 20.4 nM for human COX-2, respectively. The three NSAIDs were detected in the aqueous humor and the retinochoroidal tissue at all-time points. Simulated pharmacokinetics showed that the levels of the three NSAIDs were continuously higher than the IC50 of COX-2, as an index of efficacy, in the aqueous humor, whereas only the bromfenac concentration was continuously higher than the IC50 at its trough level in the retinochoroidal tissues. The intravitreous concentration of proteins was significantly reduced in rabbits that received topical bromfenac (P = 0.026) but not the other two NSAIDs. Conclusions Topical bromfenac can penetrate into the retinochoroidal tissues in high enough concentrations to inhibit COX-2 and exerts its inhibitory effect on the blood-retinal barrier breakdown in an experimental retinochoroidal inflammation in rabbits. Topical bromfenac may have a better therapeutic benefit than diclofenac and nepafenac for retinochoroidal inflammatory diseases.

Treatment of rabbit corneal infections with ophthalmic gatifloxacin: a concentration dependence study.

This study was designed to determine the most effective dose of gatifloxacin in ophthalmic solution for control of methicillin-resistantStaphylococcus aureus (MRSA) corneal infections in rabbits. Rabbits were inoculated by injecting 9300 colonyforming units of MRSA into the corneal stroma of the eye (n=43). They were then randomly assigned to topical administration of saline, ofloxacin 0.3%, or gatifloxacin 0.02%, 0.1%, 0.3%, or 0.5% ophthalmic solutions. Infection severity 48 hours postinoculation was assessed by masked observers using standard scales. After treatment completion, viable MRSA in corneal tissue were counted, and pathologic examinations of ocular tissues were conducted. Relative to saline, treatment with gatifloxacin 0.3% or 0.5% decreased mean infection scores at every time point from 16 to 48 hours after inoculation (P≤.012) and reduced area-underthe-curve values for infection scores by 50.3% and 54.2%, respectively (P=.00005). Rabbits treated with gatifloxacin 0.3% and 0.5% had lower areaunder-the-curve values than those treated with ofloxacin 0.3% (P≤.039). Viable MRSA in corneal tissue after gatifloxacin 0.3% or 0.5% treatment were decreased to less than 1% of those found after ofloxacin 0.3% treatment. Gram-positive colony formation and abscesses found in saline-treated corneas were distinctly alleviated by treatment with gatifloxacin 0.3% or 0.5%. No significant differences were observed between treatments with gatifloxacin 0.3% or 0.5% ophthalmic formulations and they were equally effective. Topical administration of gatifloxacin 0.3% or 0.5% ophthalmic solutions controlled MRSA corneal infections in rabbits significantly better than saline or ofloxacin 0.3%.

The Relationship of Brimonidine Concentration in Vitreous Body to the Free Concentration in Retina/Choroid Following Topical Administration in Pigmented Rabbits.

ABSTRACT Purpose: Several studies showed that repeated topical administration of brimonidine tartrate ophthalmic solution reached the human vitreous concentration above 2 nM, which is the concentration necessary to activate the α2-adrenergic receptor. The purpose of this study was to elucidate the relationship of the brimonidine concentration in the vitreous body to the free concentration in the retina/choroid which is the target site of brimonidine on neuroprotective effect after topical administration. Materials and methods: Brimonidine concentrations in the eye tissues of pigmented rabbits were determined following single ocular administration of 0.1% brimonidine tartrate ophthalmic solution at pH 7.3. Binding affinity of brimonidine to melanin and melanin content in the retina/choroid of pigmented rabbits was also examined. The concentration of free brimonidine which did not bind to melanin in the retina/choroid was calculated using the binding parameters to melanin. Results: Topically applied brimonidine rapidly distributed to intraocular tissues. The elimination rate from melanin-containing tissues such as the iris/ciliary body and retina/choroid was slower than the aqueous humor and vitreous body in pigmented rabbits. In both the anterior and posterior retina/choroid, the free brimonidine concentrations were over 100-fold lower than the total concentrations. The concentrations in the vitreous body closely matched to the free concentrations in the posterior retina/choroid. Simulated free concentrations in the posterior retina/choroid were gradually increased when 0.1% solution was instilled twice daily. Conclusion: The present data indicated that the brimonidine concentration in the vitreous body was comparable to the free concentration in the posterior retina/choroid. This suggests that the vitreous concentration can be a surrogate indicator of the free brimonidine concentration in the posterior retina/choroid. From the present findings, it is expected that multiple instillation of brimonidine tartrate ophthalmic solution may produce the sufficient free concentration for activation of the α2-adrenergic receptor in the retina/choroid in human.