Contribution of anti-inflammatory and anti-virulence effects of azithromycin in the treatment of experimental Staphylococcus aureus keratitis.

BACKGROUND: We aimed to demonstrate the contribution of anti-inflammatory and anti-virulence effects of azithromycin (AZM) in ocular surface infection treatment. METHODS: Staphylococcus aureus was injected into the corneal stroma of rabbits to induce keratitis. AZM at concentrations of 0.01, 0.1, and 1% was instilled into the eye twice daily. The eyes were examined using a slit lamp and scored. The viable bacteria in the cornea were counted at 48 h post infection. To evaluate the anti-inflammatory efficacy of AZM, S. aureus culture supernatant-induced anterior ocular inflammation in rabbit was examined using a slit lamp and scored. To evaluate the inhibitory effect of AZM on bacterial toxin production, S. aureus was cultured with AZM and hemolytic reaction in the culture supernatant was determined. RESULTS: In the bacterial keratitis model, AZM dose-dependently inhibited the increase in the clinical score. The viable bacterial count in the cornea treated with 1% AZM significantly decreased compared with that of the vehicle, whereas bacterial count in 0.01 and 0.1% AZM-treated corneas was similar to that of the vehicle. In the anterior ocular inflammation model, 0.1 and 1% AZM inhibited the increase in the clinical score. AZM inhibited hemolytic reaction at concentrations that did not inhibit bacterial growth. CONCLUSIONS: The results demonstrated that AZM has not only anti-bacterial, but also anti-inflammatory effects, and inhibits bacterial toxin production leading to ocular surface damage in bacterial infection. Thus, the therapeutic effect of AZM against ocular infections is expected to be higher than that which could be assumed if it only had anti-bacterial activity.

Development and characterization of a new rat ocular hypertension model induced by intracameral injection of conjunctival fibroblasts.

Glaucoma is a chronic optic neuropathy that leads to visual field loss. Elucidating the mechanisms underlying glaucoma is essential for developing new treatments, such as neuroprotective drugs. Various glaucoma models based on the induction of intraocular pressure (IOP) elevation have been established for use in glaucoma studies. However, the time-dependent pathological changes accompanying IOP elevation have not been fully elucidated. In this study, rat conjunctival fibroblasts were injected into the anterior chamber of rat eyes, and IOP elevation was induced for 28 days. Glaucomatous signs such as optic nerve head cupping, retinal thinning, glial activation and apoptotic signaling in the retina were obvious in the cell-injected eyes on the 14th day after injection. The pattern of retinal ganglion cell (RGC) loss differed by the magnitude of IOP elevation. The number of RGCs decreased by 37.5% in eyes with IOP lower than 50 mmHg (Under-50) and by 88.0% in those with IOP higher than 50 mmHg (Over-50) 28 days after cell injection. The RGC counts were correlated with IOP in the Under-50 group but not in the Over-50 group. Our model may contribute to the investigation of pathogenic mechanisms of glaucoma and the development of new glaucoma treatments.

Effect of Solution pH on Distribution of Ophthalmically Administered Brimonidine in Posterior Ocular Tissues in Pigmented Rabbits.

INTRODUCTION: Brimonidine bioavailability in the aqueous humor depends on the solution pH following topical administration. The purpose of this study was to investigate the effect of solution pH on brimonidine distribution in the posterior ocular tissues in pigmented rabbits. METHODS: The anterior retina/choroid, posterior retina/choroid, and vitreous body of pigmented rabbits were collected 0.67, 1.5, 3, 6, 12, 24, 168, and 360 h after the administration of a single topical dose of 0.2% brimonidine tartrate ophthalmic solution, pH 6.4 (Alphagan®; Allergan Inc., Irvine, CA, USA). Brimonidine concentrations in these tissues were quantified using liquid chromatography/tandem mass spectrometry. Pharmacokinetic parameters were determined using noncompartmental analysis, and the results were compared with tissues from eyes administered 0.1% brimonidine tartrate ophthalmic solution, pH 7.3 (Aiphagan®; Senju Pharmaceutical Co., Ltd., Osaka, Japan) in our previous study conducted using the same procedure. RESULTS: Topically applied brimonidine was distributed rapidly into the posterior tissues of the eye after a single ophthalmic administration of the 0.2% ophthalmic solution. The areas under the curve from time 0 to 360 h following dosing with the 0.2% ophthalmic solution were 500,000, 14,300, and 28.7 ng h/g in the anterior and posterior retina/choroid, and vitreous body, respectively. CONCLUSION: The differences in the areas under the curve between two ophthalmic solutions were less than the difference in drug concentrations between these two products in any tissues. This finding indicates that the change in the solution pH from 6.4 to 7.3 increases brimonidine bioavailability into the posterior ocular tissues similarly as into the aqueous humor. FUNDING: Senju Pharmaceutical Co., Ltd.

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

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.

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.

Comparison of CCD-equipped laser speckle flowgraphy with hydrogen gas clearance method in the measurement of optic nerve head microcirculation in rabbits.

The aim of this study was to verify the correlation between mean blur rate (MBR) obtained with CCD-equipped laser speckle flowgraphy (LSFG) and capillary blood flow (CBF) obtained by the hydrogen gas clearance method in rabbit optic nerve head (ONH). Using Japanese white rabbits under systemic anesthesia, a hydrogen electrode was inserted an area of the ONH free from superficial capillaries. MBR was measured with LSFG near the hydrogen electrode. CBF and MBR were measured in the range of 32.4-83.5 mL/min/100 g and 3.5-6.0, respectively. MBR and CBF were significantly correlated (r = 0.73, P < 0.01, n = 14). After inhalation of carbon dioxide (CO(2)) or intravenous administration of endothelin-1 (ET-1), MBR and CBF were changed in the relative range of 0.74-1.27 and 0.76-1.35, respectively. The relative changes in MBR and CBF induced by CO(2) and ET-1 were also significantly correlated (r = 0.67, P < 0.01). The current results suggest that MBR may correlate with CBF and also change with CBF, as an index of blood flow in the ONH, linearly.

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/mm(2) (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm(2)). 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.

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.

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 alpha (TNF-alpha) was assessed. (3) The effects of fluoroquinolones on TNF-alpha 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-alpha 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-alpha 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-alpha production at the doses used in topical ophthalmic therapy.

Effects of topical administration of y-39983, a selective rho-associated protein kinase inhibitor, on ocular tissues in rabbits and monkeys.

PURPOSE: To elucidate the intraocular pressure (IOP)-lowering effects and associated characteristics of Y-39983, a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor derived from Y-27632, in animal eyes. METHODS: Y-39983 was compared with Y-27632 for selectivity of ROCK inhibition by biochemical assay. The IOP was monitored by pneumatonometer in albino rabbits and cynomolgus monkeys that were given topically administered Y-39983. The total outflow facility and uveoscleral outflow were measured by two-level constant-pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively, at 2 hours after topical administration of Y-39983 in albino rabbits. The ocular toxicologic effects of topical administration of Y-39983 were observed in albino rabbits and cynomolgus monkeys. RESULTS: A biochemical assay showed that Y-39983 inhibited ROCK more potently than Y-27632. In rabbits, topical administration of Y-39983 significantly increased conventional outflow by 65.5%, followed by significant, dose-dependent reduction in IOP. Maximum IOP reduction was 13.2 +/- 0.6 mm Hg (mean +/- SE) at 0.1% Y-39983 in rabbits. In monkeys, at 3 hours after topical administration of 0.05% Y-39983, maximum reduction of IOP was 2.5 +/- 0.8 mm Hg. No serious side effects were observed in ocular tissues except sporadic punctate subconjunctival hemorrhage during long-term topical administration of Y-39983 four times a day (at 2-hour intervals) in rabbits or monkeys. However, punctate subconjunctival hemorrhage was not observed with administration twice daily (at a 6-hour interval) or three times a day (at 5-hour intervals). CONCLUSIONS: Y-39983 causes increased outflow facility followed by IOP reduction. Y-39983 ophthalmic solution may be a candidate drug for lowering of IOP, since it increases conventional outflow and produces relatively few side effects.

Sialic Acid in human tear fluid decreases in dry eye.

PURPOSE: To evaluate the association between tear mucin and dry eye, we compared tear fluid sialic acid concentration in dry eye patients with that in normal volunteers. We also compared the sialic acid concentration with the results of several different types of dry eye examinations. METHODS: Subjects comprised 16 age-matched, normal healthy controls and 45 dry eye patients. Dry eye examinations included fluorescein staining, rose bengal staining, Schirmer I test, cotton thread test, and fluorescein breakup time. Tear samples were collected with a micropipette after the instillation of 50 microl saline. The sialic acid concentration was measured by high-performance liquid chromatography. RESULTS: The sialic acid concentration was significantly lower in the dry eye group (5.3 +/- 4.3 microg/ml) than in the control group (37.1 +/- 28.3 microg/ml) (P < 0.001). Also, the sialic acid concentration correlated significantly with the results of dry eye examinations (P < 0.0002). CONCLUSIONS: The different sialic acid levels in the control and dry eyes may imply differences in the quantity or quality of glycoprotein in tears between these two groups. Thus, sialic acid, a component of mucin, may play a key role in the dry eye condition.

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-resistant Staphylococcus aureus (MRSA) corneal infections in rabbits. Rabbits were inoculated by injecting 9300 colony-forming 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 < or = .012) and reduced area-under-the-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 area-under-the-curve values than those treated with ofloxacin 0.3% (P < or = .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%.