Pigmented Rabbits Research Articles

Distribution of Cyclosporine A in Ocular Tissues After Topical Administration of Cyclosporine A Cationic Emulsions to Pigmented Rabbits

The aim of this study was to compare the ocular and systemic distribution of cyclosporine A (CsA) in rabbits after the instillation of preservative-free CsA cationic and anionic emulsions. For the single-dose pharmacokinetic (PK) study, rabbits were instilled with 50 μL of the test material. For the multiple-dose PK study, rabbits were instilled twice daily with Restasis or once daily with NOVA22007 for 10 days. At each time point, the cornea, conjunctiva, and whole blood were harvested for CsA quantification. Ocular and systemic distribution were determined after 4 times daily instillations with 50 μL of 3H-CsA cationic and anionic emulsions for 7 days. Restasis was used as a reference in all studies. Single-dose PK data demonstrated that NOVA22007 0.1% and 0.05% delivered higher CsA concentrations to the cornea than Restasis [concentration maximum (C max): 2692, 1372, and 748 ng/g, respectively] and have a better exposition (area under the curve). Conjunctival Cmax values were 1914, 696, and 849 ng/g and area under the curve values were 3984, 2796, and 2515 ng/g · h, for either dose of the cationic emulsions and Restasis, respectively. The multiple-dose PK and the 3H-CsA distribution data demonstrated that the systemic distribution after repeated instillations was low and comparable for all emulsions. These data demonstrate that the CsA cationic emulsions were more effective than Restasis at delivering CsA to target tissues, thus confirming the potential advantage of cationic emulsions over anionic emulsions as vehicle for ocular drug delivery for the treatment of ocular surface diseases.

Assessment of Goblet Cell Orifice Distribution Across the Rabbit Bulbar Conjunctiva Based on Numerical Density and Nearest Neighbors Analysis

Purpose: To assess density and spatial distribution of the goblet cell orifices at the surface of the rabbit bulbar conjunctiva as an indicator of functional activity.Methods: Specimens of the superior or inferior bulbar conjunctiva from six healthy young adult (2 kg) pigmented rabbits were obtained using a special preparation technique by which the conjunctiva was carefully stretched out during fixation with buffered glutaraldehyde. The apical surface of the specimens was examined by scanning electron microscopy. From high magnification prints, the areas and dimensions of 32–49 orifices/image were measured. In addition, the centre-to-centre spacing and spatial distribution of the orifices were assessed using a nearest neighbors principle.Results: The bulbar conjunctival surface is composed of polygonal cells decorated with surface microplicae and in between which are individual goblet cell orifices. The goblet cell orifices are characterized by tending to be oval in shape (long:short dimensions ratio of 1.57 +/− 0.23) and usually having a distinct line of microvilli around the perimeter. The orifices had a wide range of areas (from 13 to 188 μm2; group mean +/− SD of 54 +/− 36 μm2), and distribution of orifice areas was skewed or even bimodal. The overall orifice density was 387 +/− 68/mm2, with the group-averaged nearest neighbors distance being 34 +/− 3 μm. Comparisons of the measured nearest neighbors distances to that for an optimum spacing based on numerical density reveals the goblet cell orifices to be slightly further apart and that they were not obviously in groups or clustered.Conclusions: Goblet cell orifices at the bulbar conjunctival surface, a presumed indicator of functional secretory activity, appear to have reproducible density and a discrete and reasonably predictable spatial distribution.

Ocular pharmacokinetics of bimatoprost formulated in DuraSite compared to bimatoprost 0.03% ophthalmic solution in pigmented rabbit eyes

PurposeTo compare the aqueous humor (AH) and iris-ciliary body (ICB) concentration of bimatoprost in rabbit eyes treated with ISV-215 (0.03% bimatoprost formulated in DuraSite) with the marketed product bimatoprost 0.03% ophthalmic solution.MethodsThe left eye of rabbits received a single topical instillation of either ISV-215 (n = 32 eyes) or bimatoprost 0.03% (n = 32 eyes). At predetermined time points, levels of bimatoprost and bimatoprost acid in the AH and the ICB were quantified by HPLC-MS/MS.ResultsBoth bimatoprost and bimatoprost acid were detected in the AH and the ICB within 15 minutes of dosing. Bimatoprost acid concentrations in both compartments were markedly higher than bimatoprost. There was a statistically significant (P < 0.01) increase in the concentration of the prodrug in the AH and its acid form in the ICB in animals treated with ISV-215 compared to bimatoprost 0.03%. In the ISV-215-treated rabbit eyes, the highest concentrations of bimatoprost and bimatoprost acid were in the ICB and AH, respectively, while in the bimatoprost 0.03%-treated eyes, no differences in the drug content of the selected ocular tissues were observed.ConclusionsBimatoprost 0.03% formulated in DuraSite has superior ocular distribution and area under the curve compared to bimatoprost 0.03% in rabbit eyes. This improvement in the pharmacokinetic parameters of ISV-215 may provide us with a better platform to optimize a bimatoprost formulation that offers the same degree of efficacy in lowering intraocular pressure and improved therapeutic index in glaucomatous patients by lessening the ocular side effects associated with long-term use of topical prostaglandin F2α analogs.

Corneal stromal ablation with femtosecond ultraviolet pulses in rabbits

To determine the effectiveness of femtosecond ultraviolet (UV) pulses in ablating corneal stroma in a rabbit model and to compare the healing response between eyes treated with femtosecond UV pulses and eyes treated with standard excimer photorefractive keratectomy. Laser Research Center, Vilnius University, Vilnius, Lithuania. Experimental study. Myopic photoablation using a femtosecond UV solid-state laser system was applied to corneas of pigmented rabbits. Experiments in 16 eyes were performed for optimization of the laser system parameters (fluence, spot size, pulse repetition rate) and calibration of ablation rate. In 7 rabbits, deep femtosecond UV ablation (∼130 μm) in 1 eye and shallow ablation (∼30 μm) in the contralateral eye were performed. Nine rabbits received an approximately 30 μm ablation with femtosecond UV pulses in 1 eye and with a conventional excimer system in the contralateral eye. Two eyes were used as controls. The ablation process and surface-temperature dynamics were monitored and recorded. Surface quality and haze development were evaluated. Rabbits were humanely killed 0 to 6 months after surgery, and eyes were enucleated for histological examination. Rabbit corneas ablated with femtosecond UV pulses or excimer laser radiation were similar in terms of the corneal wound-healing process, surface quality, and histology. The experiments indicate the feasibility of clinical application of femtosecond UV lasers for stromal ablation. The ability to switch between laser harmonics allows fast changeover from infrared to the UV mode, implying that a wide range of ophthalmic procedures can be performed using a single solid-state laser device. Ms. Gabryte and Mr. Danielius are paid employees of Light Conversion Ltd. Mr. Danielius is a shareholder of Light Conversion Ltd. No other author has a financial or proprietary interest in any material or method mentioned.

Transporter Targeted Gatifloxacin Prodrugs: Synthesis, Permeability, and Topical Ocular Delivery

In this work, we aim to design and synthesize prodrugs of gatifloxacin targeting organic cation transporter (OCT), monocarboxylate transporter (MCT), and ATB (0, +) transporters and to identify a prodrug with enhanced delivery to the back of the eye. Dimethylamino-propyl, carboxy-propyl, and amino-propyl(2-methyl) derivatives of gatifloxacin (GFX), DMAP-GFX, CP-GFX, and APM-GFX, were designed and synthesized to target OCT, MCT, and ATB (0, +) transporters, respectively. An LC-MS method was developed to analyze drug and prodrug levels in various studies. Solubility and log D (pH 7.4) were measured for prodrugs and the parent drug. The permeability of the prodrugs was determined in the cornea, conjunctiva, and sclera-choroid-retinal pigment epitheluim (SCRPE) and compared with gatifloxacin using an Ussing chamber assembly. Permeability mechanisms were elucidated by determining the transport in the presence of transporter specific inhibitors. 1-Methyl-4-phenylpyridinium iodide (MPP+), nicotinic acid sodium salt, and α-methyl-DL-tryptophan were used to inhibit OCT, MCT, and ATB (0, +) transporters, respectively. A prodrug selected based on in vitro studies was administered as an eye drop to pigmented rabbits, and the delivery to various eye tissues including vitreous humor was compared with gatifloxacin dosing. DMAP-GFX exhibited 12.8-fold greater solubility than GFX. All prodrugs were more lipophilic, with the measured log D (pH 7.4) values ranging from 0.05 to 1.04, when compared to GFX (log D: -1.15). DMAP-GFX showed 1.4-, 1.8-, and 1.9-fold improvement in permeability across the cornea, conjunctiva, and SCRPE when compared to GFX. Moreover, it exhibited reduced permeability in the presence of MPP+ (competitive inhibitor of OCT), indicating OCT-mediated transport. CP-GFX showed 1.2-, 2.3-, and 2.5-fold improvement in permeability across the cornea, conjunctiva, and SCRPE, respectively. In the presence of nicotinic acid (competitive inhibitor of MCT), the permeability of CP-GFX was reduced across the conjunctiva. However, the cornea and SCRPE permeability of CP-GFX was not affected by nicotinic acid. APM-GFX did not show any improvement in permeability when compared to GFX across the cornea, conjunctiva, and SCRPE. Based on solubility and permeability, DMAP-GFX was selected for in vivo studies. DMAP-GFX showed 3.6- and 1.95-fold higher levels in vitreous humor and CRPE compared to that of GFX at 1 h after topical dosing. In vivo conversion of DMAP-GFX prodrug to GFX was quantified in tissues isolated at 1 h after dosing. The parent drug-to-prodrug ratio was 8, 70, 24, 21, 29, 13, 55, and 60% in the cornea, conjunctiva, iris-ciliary body, aqueous humor, sclera, CRPE, retina, and vitreous humor, respectively. In conclusion, DMAP-GFX prodrug enhanced solubility, log D, as well as OCT mediated delivery of gatifloxacin to the back of the eye.

Ocular Pharmacokinetics of Bevacizumab in Vitrectomized Eyes with Silicone Oil Tamponade

We characterized the ocular pharmacokinetics of bevacizumab in vitrectomized eyes with silicone oil tamponade. A total of 18 pigmented rabbits underwent vitrectomy and silicone oil tamponade before an intra-silicone oil injection of 1.25 mg bevacizumab. At post-injection days 1, 7, 14, 28, 42, and 56, 3 rabbits were sacrificed and enucleated, and bevacizumab concentrations were measured in various ocular tissues and plasma. The bevacizumab peak concentration was reached at day 14 in the aqueous humor (4030.70 ng/mL), retina (42,171.7 ng/g), and choroid (56,243.33 ng/g). In the iris/ciliary body and plasma, the peak concentration was reached at day 7 with 52,648.30 ng/g and 197.70 ng/mL, respectively. The choroid had the maximum exposure to bevacizumab with an area under the curve calculated from time zero to the last observed time (AUC(last)) of 1,151,633.40 ng/day/g and the aqueous humor had the minimum exposure (AUC(last) = 74,611.28 ng/day/g) among the ocular tissues, while the drug exposure to the plasma was the smallest of all tissues studied (AUC(last) = 3795.17 ng/day/g). The terminal half-lives and the mean residence time of bevacizumab in the ocular tissues ranged from 3-5 and 10-13 days, respectively. The peak concentration of bevacizumab in various ocular tissues and plasma was delayed and lower than that found in normal rabbit eyes; however, the terminal half-lives were similar to those found in the eyes with native vitreous following an intravitreal injection. Oil may have impacted the distribution of bevacizumab and led to an altered profile of drug level in the ocular tissues.

Miltefosine and polyhexamethylene biguanide, a new drug combination for the treatment of Acanthamoeba keratitis. Results from in‐vivo toxicological and efficacy studies

AbstractPurpose Miltefosine (MLT) and combinations of Miltefosine and polyhexamethylene biguanide (PHMB), chlorhexidine (CHX), and propamidine isethionate (PI), respectively, have been tested in a rat efficacy model for treatment of Acanthamoeba keratitis. In a second step, the most promising treatment regimens from efficacy studies have been tested for local tolerance in a fully GLP‐compliant toxicological study in pigmented rabbits.Methods The cornea of rats were infected with Acanthamoeba hatchetti. Cornea infections were graded microscopically. Nine groups were treated with varous treatment regimens for 28 days (8 times per day during the first week, and 3 times per day for the last three weeks). Efficacy of treatment was examined by determination of the degree of infection and culturing of excised eyes after the end of the treatment period. On day 28 the eyes were inspected under the microscope, re‐graded and were then compered with the grades before starting the therapy.Results Best treatment results were obtained from PHMB‐MLT group. The ratio of fully recovered eyes was 28.4%. The highets therapeutic activity was yielded by the combination MLT‐PHMB (86%), followed by MLT, CHX, or PI (72%) and the combination MLT–PI (70%. The fully GLP‐compliant study in pigmented rabbits (28 days, 8 treatments per day) showed excellent local tolerance.Conclusion Thus the combination of Miltefosine and PHMB can be seen as a highly effective and save option for treatment of AK.

Effects of tamsulosin and silodosin on isolated albino and pigmented rabbit iris dilators: Possible mechanism of intraoperative floppy-iris syndrome

To determine the mechanism of intraoperative floppy-iris syndrome (IFIS) by examining the binding affinity of tamsulosin and silodosin to α-receptors and melanin pigment using control and α(2)-blocker chronically administered in rabbit models. Department of Ophthalmology, Kitasato University School of Medicine, Kanagawa, Japan. Experimental study. The study was performed in isolated albino and pigmented rabbit iris dilators using pharmacologic and morphologic examinations. For pharmacologic examinations, the mean pK(B) values (pK(B) = -log K(B), where -log K(B) is the equilibrium dissociation constant of the antagonist-receptor complex) of tamsulosin in albino and pigmented rabbits were 9.10 and 8.08 and those of silodosin, 10.3 and 8.11, respectively. The pK(B) values of tamsulosin and silodosin in albino rabbits were significantly higher than in pigmented rabbits. In the isolated rabbit iris dilator, the maximum contraction evoked by 10(-3) mol/L phenylephrine gradually decreased by repetitive application in the chronic α-blocker-administered models. For morphologic examinations, the sizes of the pigment granules of pigment epitheliums for the α-blocker-administered models were irregular. The shape of shared nucleus of dilator muscles and pigment epitheliums changed to lobular, and the dilator muscle layer was thinner than in the control. The high affinity of α-blockers for α(1)-adrenoreceptors is important in the analysis of the mechanism of IFIS. However, IFIS should not be attributed to long-term binding with receptors alone; the drug-melanin interaction causing dilator muscle atrophy is probably the other important factor in the mechanism of IFIS.

Prediction of the Vitreal Half-Life of Small Molecular Drug-Like Compounds

To build a fast, user-friendly computational model to predict the intravitreal half-lives of drug-like compounds. We used multivariate analysis to build intravitreal half-life models using two data sets, one with experimental data derived from both pigmented and albino rabbits and another including only data from experiments with albino rabbits. The final models had a Q(2) value of 0.65 and 0.75 for the mixed and albino rabbit models, respectively. The models performed well in predicting the intravitreal half-life of an external test set. In addition, the models are physiologically interpretable, containing mainly hydrogen bonding and lipophilicity descriptors. The developed models enable reliable predictions of intravitreal half-lives for use in the early drug development stages, without the need for prior experimental data.

External sclerostomy with the femtosecond laser versus a surgical knife in rabbits.

To experimentally compare the external sclerostomy produced using a femtosecond laser with that made by a surgical knife and to evaluate the healing patterns, efficacy and technical advantages of femtosecond laser sclerostomy. In a prospective randomized, controlled, masked-observer study, 10 pigmented rabbits underwent external sclerostomy with a femtosecond laser in the right eye; 10 additional rabbits underwent sclerostomy with a surgical superblade in the right eye. Clinical characteristics, which included bleb morphology and intraocular pressure, were recorded for 1 month after surgery. Six additional rabbits underwent external femtosecond laser sclerostomy in the right eye and mechanical sclerostomy in the left eye and were killed at day 14 after surgery. Histologic staining, immunohistochemistry and scanning electron microscopy were subsequently performed to assess the morphology of the filtering fistula. The titanium-sapphire femtosecond laboratory laser was operating at a repetition rate of 1 kHz, 0.4 mJ pulse energy, a central wavelength of 800nm and a pulse duration of 50 femtoseconds. Mann-Whitney and Kaplan-Meier tests were used for statistical analysis. Successful complete sclerostomy was achieved in each laser-treated eye which was hit only once by the laser. The laser treated time was approximately 15s-16s. In the laser-treated group (n=16), 2 eyes (12%) developed mild hyphema at the site of entry and 8 eyes (50%) showed transient edema in the corneal periphery adjacent to the laser impact zone. The differences between the groups in duration of function blebs and pressure reduction were statistically significant (P=0.025 and 0.016, respectively). The success rate of the laser-treated group was significantly higher than the knife group (P=0.005). Histologically, the subconjunctival connective tissue was loosely arranged with partially patent sclerostomy in the laser-treated eyes at postoperative day 14. This contrasted with the completely scarred sclerostomy tract in the knife group. The mean numbers of fibroblasts and new vessels as well as the amount of new collagen deposition at bleb site were significantly decreased in the laser group (P=0.045, 0.013 and 0.036, respectively). The current study demonstrates that external femtosecond laser sclerostomy may offer a safe and effective alternative for the minimally invasive surgical management of glaucoma.

Evaluation of extended release brimonidine intravitreal device in normotensive rabbit eyes

To evaluate the safety profile of a brimonidine extended release intravitreal implant, in normotensive rabbit eyes. Devices were made from hollow poly-l-lactic acid (PLA) tubes and contained hundred micrograms of brimonidine pamoate. Device was injected intravitreally in one eye of 12 New Zealand pigmented rabbits, whereas other eye was injected with a sham implant in masked fashion. Ocular examination was conducted at baseline and months 1, 3 and 6 including dilated fundus examination and electro-retinogram (ERG). Four rabbits were sacrificed at each time-point for retinal histology. ERG data were compared between groups and time-points using anova. No complications were reported from either eye of any rabbits over a 6-month period. Photopic A wave was reduced in the control eye at 1 month compared with baseline (p < 0.01). There was no significant difference in other ERG parameters between the groups at different time-points. Gross retinal histology was normal at all time-points. Extended release intravitreal brimonidine device was found to be safe and in normotensive rabbit eyes.

Normal corneal thickness measurements in pigmented rabbits using spectral‐domain anterior segment optical coherence tomography

To document the thickness of the central cornea in pigmented rabbits using spectral-domain anterior segment optical coherence tomography (AS-OCT). Seventeen pigmented rabbits (6 male, 11 female, both eyes) were involved in this study. Thirty-four eyes from healthy pigmented rabbits underwent a complete ophthalmologic examination, including AS-OCT. Eight radial scans, 6 mm in length and centered on the cornea, were obtained using the AS-OCT. Corneal thickness was automatically calculated using pachymetry software. Measurements were displayed as the mean and standard deviation for each of the 17 regions defined by the software. The regions were the center (1 mm radius, area a), the inner ring (2.5 mm radius, area b), the outer ring (3 mm radius, area c), and the eight radial scan lines in eight directions (Superior (1), SN (2), Nasal (3), IN (4), Inferior (5), IT (6), Temporal (7), ST (8)) with an angle of 45° between each consecutive scan line (a, b 1-8, c 1-8). There was no statistically significance difference in corneal thickness between gender, eye, and the eight directions (P = 0.804, P > 0.05, P > 0.05). There was a statistical difference between the thickness in areas a, b, and c (P < 0.05). The corneal thickness increased gradually from the center to the periphery of the 6 mm measured. The center corneal thickness was 387 ± 19.8 μm for OD and 384 ± 20.2 μm for OS. The corneal thicknesses of the thinnest point of the right eyes (OD) and the left eyes (OS) were 376 ± 20.2 and 370 ± 16.8 μm, respectively. There was positive correlation between the thinnest point and area a in both the right eyes (r = 0.892, P < 0.001) and the left eyes (r = 0.832, P < 0.001). This is the first documentation of the rabbit corneal thickness in vivo using the spectral-domain AS-OCT. Pigmented rabbit corneas were almost 150 μm thinner than human corneal values. Gender and eye were not associated with any statistical differences in central corneal thickness in this study.

Effects of 7-methylxanthine on form-deprivation myopia in pigmented rabbits.

To determine the effect of 7-methylxanthine (7-MX) on the posterior sclera of form-deprivation myopia (FDM) in pigmented rabbits. Sixteen pigmented rabbits were monocularly deprived (MD) by suturing the right eyelids after natural eye opening (ten-day old) for a period of 30 days. Two groups of pigmented rabbits were fed either 7-MX (30 mg per kg body weight; n=8) or vehicle control (saline equal volume with 7-MX; n=8). Ocular refractions, axial lengths and body weights were measured at the start and the end of the experiment 30 days later. Electron microscopy was used to measure and determine the collagen fibril diameters in the posterior pole of sclera. In vehicle control MD pigmented rabbits, 30 days of MD produced -1.10D±0.78D of myopia and the axial length increased 0.51mm±0.09mm. In MD pigmented rabbits fed with 7-MX, 30 days of MD induced only -0.21D±0.11D of myopia and the axial length increased 0.07mm±0.10mm. There was significant change in axial length of vehicle control MD pigmented rabbits (13.11mm±0.19mm versus 12.60mm±0.06mm; P=0.03). The changes in refraction and axial length of two MD groups' contralateral eyes during the 30 days were not significantly different (2.75D±0.27D versus 2.75D±0.35D, P>0.05; 12.60mm±0.06mm versus 12.45mm±0.14mm, P>0.05). The weights of the two groups pigmented rabbits had no significant changes (187g±22.1g versus 189g±19.3g, P>0.05). The diameter of scleral collagen fibers increased in both eyes of 7-MX treated pigmented rabbits. There was significant difference in collagen fibril diameters of inner layer (111.34nm±28.30nm versus 94.80nm±27.52nm, P=0.002) and outer layer (167.92nm±55.82 nm versus 144.04 nm±47.02nm, P=0.016) in the posterior sclera between the myopic eyes of vehicle control MD group and contralateral eyes of 7-MX treated MD group. 7-MX appears to prevent FDM in pigmented rabbits by remodeling the posterior sclera.

Role of Vascular Endothelial Growth Factor in the Breakdown of the Blood-Aqueous Barrier After Retinal Laser Photocoagulation in Pigmented Rabbits

Retinal laser photocoagulation is used to treat a variety of retinal diseases. Breakdown of the blood-aqueous barrier has been noted after retinal laser photocoagulation. The effect of vascular endothelial growth factor (VEGF) on the function of the blood-aqueous barrier after retinal laser photocoagulation remains undetermined. The current study was designed to evaluate the relationship between intraocular levels of VEGF and breakdown of the blood-aqueous barrier after retinal laser photocoagulation in rabbits. Pigmented rabbits were treated with retinal laser photocoagulation in one eye; the other served as control. Laser flare photometry was carried out on post-treatment days 1, 3, 7, and 14. Animals were sacrificed at the time period just mentioned postlaser, the eyes were removed, and samples of vitreous and aqueous humor were collected. Intraocular VEGF levels were measured by using an immunoassay. An intravitreal injection of VEGF was administered, and the aqueous flare intensity and VEGF levels in the aqueous and vitreous humor were measured at the time periods just mentioned. A significant increase in the aqueous flare intensity after retinal laser photocoagulation was noticed on postoperative day 1, with the values returning to baseline levels on day 14. The VEGF levels in the vitreous of the lasered eyes were significantly increased on day 1 compared with the nonlasered control eyes. The VEGF levels in the aqueous humor of the lasered eyes were also significantly increased on day 1 compared with the control eyes. An intravitreal injection of VEGF induced a significant increase in the aqueous flare intensity and VEGF levels in the aqueous and vitreous humor. The current results suggested that retinal laser photocoagulation can produce a breakdown of the blood-aqueous barrier. VEGF may play a role in the blood-aqueous barrier dysfunction after retinal laser photocoagulation.

A Simultaneous Iris Angiography Technique in Pigmented Rabbits

Purpose: To report a simultaneous iris angiography (IA) technique combined with the use of indocyanine green (ICG) and fluorescein sodium (FS) in pigmented rabbits. Methods: 15 rabbits were randomly divided into 3 groups according to the dye doses: the dose of the lower-volume group was 2 mg FS and 2 mg ICG; the dose of the moderate-volume group was 5 mg FS and 5 mg ICG, and the dose of the higher-volume group was 8 mg FS and 8 mg ICG. Fifteen IAs were performed by a confocal scanning laser ophthalmoscope and a rapid localization technique giving a flat-on image. Results: Simultaneous digital angiography provided two kinds of a fully dynamic video of iris vessels fluorescein in all pigmented rabbits, which is the same in pigmented and albino rabbits and no vascular pattern was hidden by the iris stromal pigment. Furthermore, dye doses influenced the IA effect in rabbits. The lower-volume dye can demonstrate iris vessels, without obvious leakage, which, however, extinguish rapidly after 10–15 s. In the higher-volume group, vascular imaging lasted longer, but subsequently significant FS and ICG leakage appeared as streams on the surface of the iris until the dye disappeared from the iris vessels; the diffuse and intense aqueous fluid lasted 1 day. The moderate-volume dye displayed vessels clearly by ICG for 300 s, without leakage; FS clear vessels were maintained for about 15–20 s and the structure of the iris became fuzzy due to quick leakage. Conclusions: Simultaneous IA with a rapid localization technique allows high-quality imaging of the pigmented rabbits and the moderate dosage of 5 mg FS and 5 mg ICG is preferred for best visualization.

Novel ophthalmic timolol meleate liposomal-hydrogel and its improved local glaucomatous therapeutic effect in vivo

To overcome the limitations of common eye drops, the study developed a novel timolol mealate (TM) liposomal-hydrogel to enhance drug permeability and prolong residence time in the precorneal region, which achieved more effective local glaucomatous therapeutic effect. Firstly, TM liposome was prepared by an ammonium sulfate gradient-pH regulation method, which its entrapment efficiency reached up to 94% and its averaged particle size is 187 nm with narrow distribution. The corneal permeability through isolated rabbit cornea was measured by modified Franz-type diffusion cells. The results of trans-corneal penetration exhibited that the apparent permeability coefficients (Papp) and the flow rates of steady state (Jss) of TM liposome was 1.50-fold higher than that of the commercialized eye drop, while TM liposome with 0.02% transcutol P was 2.19 times. In order to increase the retention time and improve the stability of liposome, we further developed a TM liposomal-hydrogel formulation by adding 1.0% HPMC K4M in TM liposome. The results showed an stability during a 120 days storage period than TM liposome. Precorneal retention study in vivo indicated that the optimal liposomal-hydrogel formulation had improved bioavailability and its retention time on rabbit corneal surface were significantly longer than that of pure liposomes or eye-drops. No obvious irritations to rabbit eyes were observed by histopathology microscopy after 7 days exposure.. Comparing to the eye drops, the TM liposomal-gel displayed prolonged therapeutic effect in cornea and greatly lowered the intraocular pressure IOP on the eyes of normal and glaucomatous pigmented rabbits.

A possible mechanism for transpupillary thermotherapy: nitric-oxide-related cellular damage.

To determine the oxidative stress markers on rabbit vitreous following transpupillary thermotherapy (TTT) application. The pigmented rabbit eyes were divided into 3 groups, each containing 6 eyes. Group 1 was used as a control group. Twelve eyes underwent TTT with a power of 250 mW (group 2) and 800 mW (group 3), with a diameter of 3000 microm and duration of 60 s; 24 h after laser application, vitreous samples were collected. Nitric oxide (NO) and malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities were determined in all groups. NO levels were statistically significantly higher in all groups when compared with the control group (p < 0.05). MDA levels were higher and SOD activities were lower in all groups when compared with the control group, but the differences were not significant statistically (p > 0.05). Our results support the hypothesis that TTT application induces NO synthesis, which may lead to occlusion at choroidal neovessels. Because of the nonsignificantly increased levels of MDA and decrease of SOD activities there maybe only a weak relation between lipid peroxidation induced by free oxygen radicals and TTT-induced vascular damage.

Ocular Pharmacokinetics of Dorzolamide and Brinzolamide After Single and Multiple Topical Dosing: Implications for Effects on Ocular Blood Flow

Ophthalmic carbonic anhydrase inhibitors have been shown to improve retinal and optic nerve blood flow. However, the relative tissue distributions of commercially available carbonic anhydrase inhibitors to the optic nerve are not known. The objective of this study was to compare the ocular pharmacokinetics and tissue distribution profiles of dorzolamide and brinzolamide after single and multiple topical applications. Pigmented rabbits were treated with single or multiple topical administrations of 30 μl of Trusopt (dorzolamide hydrochloride ophthalmic solution, 2%) to one eye and 30 μl of Azopt (brinzolamide ophthalmic suspension, 1%) to the other eye. Rabbits were euthanized at 10 predetermined time intervals over a period of 24 h, and ocular tissues and plasma samples were collected. For multiple dosing, rabbits were dosed twice per day with an 8-h interval between two doses, groups of rabbits were euthanized at 7, 14, and 21 days at 1 h after the last dose, and ocular tissues and plasma samples were collected. Drug levels in tissue samples were measured using liquid chromatography/tandem mass spectrometry. Pharmacokinetic parameters (C(max), T(max), and AUC(0-24)) were estimated by noncompartmental analysis. After a single dose, dorzolamide delivery (AUC(0-24)) to the aqueous humor, anterior sclera, posterior sclera, anterior retina, posterior retina, anterior vitreous, and optic nerve was 2-, 7-, 2.6-, 1.4-, 1.9-, 1.2-, and 9-fold higher than those of brinzolamide. C(max) was 2- to 5-fold higher for dorzolamide than that of brinzolamide in all of the ocular tissue. After multiple dosing, dorzolamide levels in the aqueous humor, sclera, retina, vitreous humor, and optic nerve were higher than those of brinzolamide, but statistical significance was achieved only with aqueous humor, vitreous humor, and optic nerve. Dorzolamide levels in the aqueous humor, anterior vitreous, posterior vitreous, and optic nerve were 1.4- to 3.2-, 2.4- to 2.7-, 2.2- to 4.5-, and 2.4- to 5.2-fold higher than those of brinzolamide. Upon multiple dosing, both drugs accumulated in all of the tissues except the conjunctiva, where the drug levels were lower than those observed with single dosing. No significant differences were found in the AUC values of these two drugs in the cornea and conjunctiva after single and multiple dosing. Drug levels were significantly higher in anterior regions than posterior regions in the sclera, retina, and vitreous for both drugs.

Modified β-Cyclodextrin (SBE7-β-CyD) with Viscous Vehicle Improves the Ocular Delivery and Tolerability of Pilocarpine Prodrug in Rabbits

The complexation of pilocarpine prodrug, O,O'-dipropionyl-(1,4-xylylene) bispilocarpate, with various beta-cyclodextrin (beta-CyD) derivatives was studied by the phase solubility method. The effects of coadministered sulphobutyl ether beta-CyD (SBE7-beta-CyD) with and without poly(vinyl alcohol) (PVA) on the miotic response and eye irritation of the prodrug were investigated in pigmented rabbits. The pilocarpine prodrug formed 1:1 inclusion complexes with variably substituted sulphobutyl ether derivatives of beta-CyD (SBE4-beta-CyD and SBE7-beta-CyD), and 1:1 and 1:2 complexes with hydroxypropyl-beta-CyD (HP-beta-CyD) at pH 7-4. Coadministered SBE7-beta-CyD eliminated the eye irritation due to the pilocarpine prodrug, but also decreased the miotic response. Ocular absorption of the prodrug was improved by increasing the viscosity of prodrug/SBE7-beta-CyD solution with PVA without inducing any eye irritation. Eye irritation due to viscous prodrug/SBE7-beta-CyD solutions was comparable with isotonic NaCl solution. We conclude that administration of pilocarpine prodrug in viscous SBE7-beta-CyD solution decreases substantially eye irritation while ocular absorption is not affected.

A comparative study on ocular damage induced by 1319nm laser radiation

High energy loss of 1,319 nm laser due to pre-retinal water absorption makes the ocular axial length more critical, while the relative low absorbance of melanin makes retinal pigmented epithelium less contributing, to retinal damage threshold. However, both have never been illustrated experimentally. Here we determined and compared the retinal damage thresholds at this wavelength in three species with different axial lengths and retinal pigmentations. The corneal damage threshold was also determined for further comparative analysis. The retinal damage thresholds of albino rat, non-pigmented and pigmented rabbit, and the corneal damage threshold of non-pigmented rabbit were determined for 0.2 and/or 0.4-second exposure durations. The incident beam diameter on cornea was 5 mm for rabbit retinal and 2 mm for rat retinal and rabbit corneal lesion. Minimum visible lesions were examined 1- and 24-hour post-exposure. Probit analysis was used to establish the estimated damage threshold for 50% of exposures (ED(50) ). The direct transmittance of pre-retinal eye media was used for further comparative analysis. The retinal ED(50) of albino rat for 0.2 seconds, non-pigmented rabbit for 0.2 seconds, 0.4 seconds, pigmented rabbit for 0.4 seconds was 8.8, 12.1, 22.5, 18.5 J/cm(2) (0.28, 2.4, 4.4, 3.6 J in total intraocular energy (TIE)). The corneal ED(50) was 86.1 J/cm(2) . Under the condition of 5 mm beam diameter and 0.4-second exposure duration, the calculated retinal ED(50) of human was higher, while that of rhesus monkey was lower than the corneal ED(50) . For 1,319 nm laser radiation, the ocular axial length has great, while the retinal pigmentation has only slight influence on retinal damage threshold. The relative lower direct transmittance of human eye media makes the retina more resistant to laser lesion, which should be considered when deriving human maximum permissible exposure (MPE).