Ocular and systemic pharmacokinetics of brimonidine and brinzolamide after topical administration in rabbits: comparison between fixed-combination and single-drug formulations.

AIM: The aim of this study was to compare the ocular and systemic absorption of brimonidine (BMD) and brinzolamide (BZM) in rabbits after the topical administration of a fixed-combination ophthalmic suspension of 0.1% BMD tartrate and 1% BZM (FCBB) with that after the administration of the respective single-drug formulations. MATERIALS AND METHODS: Ocular and systemic drug absorption was estimated by determining BMD and BZM concentrations in the aqueous humor, retina/choroid, vitreous body, and blood/plasma by liquid chromatography/tandem mass spectrometry after the administration of FCBB, 0.1% BMD tartrate ophthalmic solution (0.1% BMD), or 1% BZM ophthalmic suspension (1% BZM) to rabbits. RESULTS: In concomitant administration, instilling 0.1% BMD and 1% BZM successively without interval lowered aqueous humor concentrations of both drugs compared to those observed with a 5-min interval. After FCBB administration, BMD and BZM concentrations in the aqueous humor were comparable with those observed after the administration of 0.1% BMD and 1% BZM, whereas BMD concentrations in posterior ocular tissues were equal to or higher than those observed after 0.1% BMD. Plasma BMD concentrations following the administration of FCBB were 0.8-fold lower than those after 0.1% BMD; no remarkable differences were observed in blood BZM concentrations for both formulations. CONCLUSIONS: FCBB achieved drug distribution in the aqueous humor and systemic exposure that were comparable to those for the single-drug formulations. The viscosity of FCBB may increase BMD distribution in the retina/choroid. The administration interval affects ocular drug absorption with the concomitant administration of 0.1% BMD and 1% BZM, which can be overcome by using the fixed-combination of both drugs.

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.

Ocular and Systemic Pharmacokinetics of Brimonidine and Timolol After Topical Administration in Rabbits: Comparison Between Fixed-Combination and Single Drugs.

INTRODUCTION: This study was aimed to compare ocular tissue distribution and systemic exposure of brimonidine and timolol after single topical administration to rabbits of fixed-combination ophthalmic solution of 0.1% brimonidine tartrate and 0.5% timolol and single drugs (0.1% brimonidine tartrate ophthalmic solution or 0.5% timolol ophthalmic solution) or concomitant administration of single drugs. METHODS: Rabbits were treated with a single topical administration of each ophthalmic solution or concomitant administration of single drugs. For concomitant administration, 0.1% brimonidine tartrate was administered after 0.5% timolol instillation successively within 10 s (without interval) or with 5-min intervals. Brimonidine and timolol concentrations in the aqueous humor, retina/choroid, vitreous body, and plasma were determined with liquid chromatography-tandem mass spectrometry. RESULTS: The area under the curve values of both drugs in the aqueous humor after fixed-combination administration were comparable to those after concomitant administration. The value of brimonidine was comparable to that after 0.1% brimonidine tartrate administration, whereas the value of timolol was 1.6-fold higher than that after 0.5% timolol administration. The plasma area under the curve value of brimonidine did not differ between fixed-combination and single-drug administrations, but that of timolol was higher after fixed-combination administration than after single-drug administration. Similar concentration-time curves of brimonidine were observed in the posterior ocular tissues in all groups. For concomitant administration, both drug concentrations in the aqueous humor without an administration interval were lower than those with 5-min intervals. CONCLUSION: There was no difference in the effect of formulation compositions on ocular and systemic pharmacokinetics among the ophthalmic solutions, but brimonidine may alter the ocular and systemic absorption of timolol, which is possibly due to its pharmacologic action. We demonstrated the importance of an administration interval in the concomitant administration of these drugs. This concern could be avoided by using a fixed combination of brimonidine and timolol.

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.

In Silico Ocular Pharmacokinetic Modeling: Delivery of Topical FK962 to Retina.

PURPOSES: To establish the in silico ocular pharmacokinetic modeling for eye drops, and to simulate the dose regimen for FK962 in human choroid/retinal diseases. METHODS: Pharmacokinetics for FK962 in vivo was performed by a single instillation of drops containing 0.1% 14C-FK962 in rabbit eyes. Permeation of FK962 across the cornea, sclera, and choroid/retina was measured in vitro. Neurite elongation by FK962 was measured in cultured rat retinal ganglion cells. Parameters from the experimental data were used in an improved in silico model of ocular pharmacokinetics of FK962 in man. RESULTS: The mean concentration of FK962 in ocular tissues predicted by in silico modeling was consistent with in vivo results, validating the in silico model. FK962 rapidly penetrated into the anterior and posterior segments of the eye and then diffused into the vitreous body. The in silico pharmacokinetic modeling also predicted that a dose regimen of 0.0054% FK962 twice per day would produce biologically effective concentrations of FK962 in the choroid/retina, where FK962 facilitates rat neurite elongation. CONCLUSIONS: Our in silico model for ocular pharmacokinetics is useful (1) for predicting drug concentrations in specific ocular tissues after topical instillation, and (2) for suggesting the optimal dose regimens for eye drops. The pharmacodynamics for FK962 produced by this model may be useful for clinical trials against retinal neuropathy.

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.

Vitreous and aqueous concentrations of brimonidine following topical application of brimonidine tartrate 0.1% ophthalmic solution in humans.

PURPOSE: To determine the vitreous and aqueous concentrations of brimonidine after topical application of the ophthalmic solution 0.1%. METHODS: The prospective observational case series included patients with an idiopathic epiretinal membrane or macular hole who were scheduled for a pars plana vitrectomy. Brimonidine tartrate ophthalmic solution 0.1% was topically administered twice daily for 1 week preoperatively. Vitreous and aqueous humor was collected before vitrectomy, and then, the brimonidine concentration was measured with liquid chromatography tandem spectrometry (LC/MS/MS). RESULTS: Twenty-four patients (19 phakic eyes and 5 pseudophakic eyes) were enrolled. The mean concentrations in the aqueous humor and vitreous were 336.0 ± 276.2 and 4.8 ± 3.2 nM, respectively. A significant relationship was observed between the vitreous and aqueous samples (P = 0.034, R(2) = 0.22). Nineteen (79%) of the 24 eyes showed more than 2 nM of brimonidine tartrate concentration in the vitreous. In the phakic eyes, the mean concentration of brimonidine in the vitreous was 4.9 ± 3.3 nM, while the mean concentration in the pseudophakic eyes was 4.1 ± 2.4 nM, demonstrating no significant difference between pseudophakic and phakic eyes (P = 0.59). CONCLUSIONS: After 1 week of dosing, in most of the patients who topically received brimonidine tartrate 0.1%, the concentration in the vitreous of the molecule was above 2 nM, which is known to activate neuroprotective α-2 receptors in animal retina. The drug penetration into the vitreous seems to be independent of lens status.

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.

Effect of dosing interval on the efficacy of topical ophthalmic gatifloxacin against Enterococcus faecalis in an in vitro pharmacokinetic model simulating the local eye compartment.

Improved dosing regimens have been proposed for various antimicrobial agents by application of pharmacokinetic/pharmacodynamic (PK/PD) principles. However, for topical ophthalmic use there are several limitations to changing the dosing regimen, such as drug formulation and bioavailability. In this study, we investigated the relationship between dosing interval and antibacterial efficacy in an in vitro PK model mimicking post-operative endophthalmitis. The in vitro PK model simulated the aqueous humour concentration following topical application of 0.3% gatifloxacin ophthalmic solution to rabbit eyes. A clinical isolate of Enterococcus faecalis was exposed to gatifloxacin three times repeatedly at various intervals from 0 h to 8 h. The area between the control growth curve and the bacterial killing and re-growth curve for 24 h (ABBC) was used to evaluate efficacy. The ABBC showed bell-shaped dependence on the dosing interval with a peak at 3h. Under limited condition of total exposure amount, i.e. area under the concentration-time curve, the antimicrobial efficacy appears to be associated with the cumulative time of a 24-h period such that the concentration exceeds the minimum inhibitory concentration (T>MIC) rather than the peak concentration:MIC ratio. The length of intermission of T>MIC during repeated dosing appears to be proportional to the decrease in efficacy of gatifloxacin against E. faecalis. A longer dosing interval, as long as T>MIC is continuous, would likely be more efficient at preventing post-operative enterococcal endophthalmitis. However, further investigation is necessary to explore whether this model is applicable to a variety of pathogens and drugs.

Contribution of secreted proteases to the pathogenesis of postoperative Enterococcus faecalis endophthalmitis.

PURPOSE: To determine how a secreted protease contributes to the pathogenesis of post-cataract endophthalmitis caused by Enterococcus faecalis using an aphakic rabbit endophthalmitis model. SETTING: Department of Ophthalmology, Ehime University School of Medicine, Ehime, Japan. METHODS: The pathogenesis of E faecalis OG1S (secreted protease-positive) and E faecalis OG1X (secreted protease-negative derivative of OG1S) was compared. After lens removal by phacoemulsification, either strain was inoculated into the lens bag. Changes in bacterial growth, electroretinography (ERG), and pathology of eyes were comparatively monitored throughout the course of the infection. Alternatively, culture fluid from either strain was injected into the vitreous body and ERG and pathology of the eyes were also examined. RESULTS: The levels of growth in the anterior chamber and vitreous cavity were similar for both strains. However, infection with OG1S resulted in a significantly greater reduction in ERG b-wave amplitude than OG1X. Histological examination showed that the posterior lens capsules were severely affected in eyes infected with OG1S, and inflammatory cells and cocci were found in the anterior vitreous cavity 24 hours after the infection. By 48 hours, the retina architecture was profoundly affected in eyes infected with OG1S. In contrast, few pathological changes were noted in the posterior lens capsules and retina of eyes infected with OG1X. Culture fluid in which OG1S had grown decreased ERG b-wave amplitude and caused morphological changes of the posterior capsule and retina similar to those in the infected eye. CONCLUSION: An extracellular protease plays a major role in the pathogenesis of E faecalis-induced postoperative endophthalmitis.

Prophylactic efficacy of ophthalmic quinolones in experimental endophthalmitis in rabbits.

PURPOSE: The aim of this study was to determine the efficacy of 0.3% gatifloxacin ophthalmic solution in preventing bacterial endophthalmitis in rabbits. METHODS: Eighty-four (84) albino phakic rabbits were injected unilaterally with 2 x 10(4) colony forming units of Enterococcus faecalis into the anterior chamber. The eyes received 0.3% ofloxacin ophthalmic ointment or 0.3% gatifloxacin ophthalmic solution with different regimens in three separate experiments: (1) 1 or 3 drops of gatifloxacin every 2 h or a single application of ofloxacin for 1 day; (2) 3 drops/day of gatifloxacin application started at 0, 6, and 24 h postinoculation, or 1 drop at 0 h, and 3 times daily gatifloxacin for the following 3 days; and (3) 1 or 3 drops of gatifloxacin application started at 0 h and no further application for the following 3 days. The control eyes received no treatment in the three experiments. The effectiveness of these different regimens was assessed by slit-lamp biomicroscopy and bacterial colony counts. The ocular penetration of the drugs was determined in a separate experiment, using 36 normal albino rabbits. RESULTS: The concentration-time curves for gatifloxacin and ofloxacin appeared parallel, with mean peak concentrations of 1161 and 219 ng/mL, respectively, at 1 h postinstillation. In Experiment 1, gatifloxacin significantly reduced the inflammation and the number of living bacteria in the aqueous humor, compared with controls, whereas ofloxacin ointment did not. A single application of ofloxacin ointment was not better than 1 drop of gatifloxacin. The results of Experiment 2 showed that the effectiveness of gatifloxacin decreased as the interval between the inoculation and the onset of treatment increased. In Experiment 3, only 3 drops of gatifloxacin on day 1 kept the inflammation significantly lower than that in the control for 4 days. CONCLUSIONS: Immediate postoperative prophylaxis would likely be effective in reducing the risk of enterococcal endophthalmitis by topical gatifloxacin.