Dutch-belted Rabbits Research Articles

Low-Level Primary Blast Causes Acute Ocular Trauma in Rabbits.

The objective of this study was to determine whether clinically significant ocular trauma can be induced by a survivable isolated primary blast using a live animal model. Both eyes of 18 Dutch Belted rabbits were exposed to various survivable low-level blast overpressures in a large-scale shock tube simulating a primary blast similar to an improvised explosive device. Eyes of the blast-exposed rabbits (as well as five control rabbits) were thoroughly examined before and after blast to detect changes. Clinically significant changes in corneal thickness arose immediately after blast and were sustained through 48 h, suggesting possible disruption of endothelial function. Retinal thickness (RT) increased with increasing specific impulse immediately after exposure. Intraocular pressure (IOP) was inversely correlated with the specific impulse of the blast wave. These findings clearly indicate that survivable primary blast causes ocular injuries with likely visual functional sequelae of clinical and military relevance.

Retinal Toxicity of Intravitreal Polyethylene Glycol 400

Polyethylene glycol (PEG)-400 is a clear, colorless viscous liquid that readily dissolves many hydrophobic drugs. It is a widely used commercial solvent and vehicle for many pharmaceutical and cosmetic products. The purpose of this study is to investigate the intravitreal safety of PEG-400 to determine its suitability as a potential vehicle for intraocular drug delivery. Six healthy, male, Dutch Belted rabbits, weighing between 2.0 and 3.0 kg, were used for this experiment. The left eye of each animal received a single 0.1 mL intravitreal injection of PEG-400. Complete eye exams and dark- and light-adapted electroretinograms (ERG) were obtained at baseline and at 1 and 4 weeks after injection. After the 4-week ERG was completed, animals were euthanized, eyes were enucleated, and histology and transmission electron microscopy (TEM) were performed. Ophthalmic examinations demonstrated diffuse signs of retinal degeneration and cataract formation in all 6 eyes injected with PEG-400, which was apparent at 1 week, but more evident by 4 weeks. Photopic and scotopic ERG studies demonstrated significant reduction in amplitudes at 1 and 4 weeks after injection compared to baseline. Histopathological and TEM analysis of eyes demonstrated both inner and outer retinal atrophy. Intravitreal injection of PEG-400 resulted in severe ocular toxicity.

Comparative assessment of ocular toxicity endpoints following intravitreal injection versus ocular instillation in Dutch-Belted rabbits

Comparative assessment of ocular toxicity endpoints following intravitreal injection versus ocular instillation in Dutch-Belted rabbits

Evaluation of a 345 nm Femtosecond Laser for Corneal Surgery with Respect to Intraocular Radiation Hazard.

PurposeWe report our findings from a preclinical safety study designed to assess potential side effects of corneal ultraviolet femtosecond laser treatment on lens and retina.MethodsRefractive lenticules (-5 dpt) with a diameter of 6 mm were created in the right cornea of eight Dutch Belted rabbits. Radiant exposure was 0.5 J/cm² in two animals and 18 J/cm² in six animals. The presence of lens opacities was assessed prior to and up to six months following laser application using Scheimpflug images (Pentacam, Oculus) and backscatter analysis (Opacity Lensmeter 702, Interzeag). Ganzfeld flash and flicker electroretinogram (ERG) recordings were obtained from both eyes prior to and up to six weeks following laser application. At the study endpoint, retinas were examined by light microscopy.ResultsIndependent of energy dose applied, no cataract formation could be observed clinically or with either of the two objective methods used. No changes in ERG recordings over time and no difference between treated and untreated eye were detected. Histologically, retinal morphology was preserved and retinal pigment epithelium as well as photoreceptor inner and outer segments appeared undamaged. Quantitative digital image analysis did not reveal cell loss in inner or outer nuclear layers.ConclusionsOur analysis confirms theoretical considerations suggesting that ultraviolet femtosecond laser treatment of the cornea is safe for intraocular tissues. Transmitted light including stray light induces no photochemical effects in lens or retina at energy levels much higher than required for the clinical purpose. These conclusions cannot be applied to eyes with pre-existing retinal damage, as these may be more vulnerable to light.

Ocular Hypotensive Activity of a Non-Peptide Bradykinin B2-Receptor Antagonist (WIN-64338) In Dutch-Belt Rabbits- A Case of Poly-Pharmacology in Action

Ocular Hypotensive Activity of a Non-Peptide Bradykinin B2-Receptor Antagonist (WIN-64338) In Dutch-Belt Rabbits- A Case of Poly-Pharmacology in Action

Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure

Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 h post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME.

Effect of AR-13324 on episcleral venous pressure in Dutch belted rabbits.

Purpose: AR-13324 is a potential new drug for the treatment of patients with glaucoma that has been shown to lower intraocular pressure (IOP) by increasing trabecular outflow facility and decreasing aqueous production. The present study tested the hypothesis that AR-13324 also lowers IOP by reducing episcleral venous pressure (EVP).Methods: In Dutch Belted (DB) rabbits (n=11), arterial pressure (AP), IOP, carotid blood flow (BFcar), heart rate (HR), and EVP were measured invasively. Animals were dosed with AR-13324 (0.04%, topical, n=6) once daily for 3 days. On day 3, the animals were anesthetized, and then, measurements were obtained before dosing with AR-13324 or vehicle (n=5) and for 3 h after dosing. The data (mean±standard error of the mean) were analyzed by repeated measures ANOVA with post hoc testing. Retrospective baseline data from prior similar studies in New Zealand White rabbits were also compiled.Results: Baseline values were as follows: AP, 101±3 mmHg; IOP; 33±3 mmHg; EVP, 16±1 mmHg; BFcar, 41±4 mL/min; and HR, 330±6 bpm. Three hours after AR-13324 dosing, IOP was reduced by 39%±7% (P<0.001) and EVP decreased by 35%±4% (P<0.05); after vehicle dosing, IOP was reduced by 24%±4% (P<0.05) and EVP increased by 25%±5% (P<0.05). AP, BFcar, and HR were unchanged.Conclusions: AR-13324 produces statistically significant lowering of EVP in DB rabbits. In addition, the baseline values for AP, IOP, EVP, BFcar, and HR in the DB rabbit are higher than those previously reported in the New Zealand rabbit.

Neurostimulation of the lacrimal nerve for enhanced tear production.

To design a proof-of-concept study to assess the effect of lacrimal nerve stimulation (LNS) with an implantable pulse generator (IPG) to increase aqueous tear production. Experimental animal study design of 6 Dutch Belted rabbits. Ultra high-resolution optical coherence tomography (UHR-OCT) quantified tear production by measuring the baseline tear volume of each rabbit's OD and OS. A neurostimulator was implanted adjacent to the right lacrimal nerve. After 2 minutes of LNS (100 μs, 1.6 mA, 20 Hz, 5-8 V), the tear volumes were measured with UHR-OCT. The change in tear volume was quantified and compared with the nonstimulated OS. Three rabbits underwent chronic LNS (100 μs, 1.6 mA, 10 Hz, 2 V) and their lacrimal glands were harvested for histopathologic analysis. The UHR-OCT imaging of the OD tear volume showed a 441% average increase in tear production after LNS as a percent of baseline. After stimulation, OD had statistically significant greater increase in tear volumes than OS (p = 0.028, Wilcoxon test). Poststimulation OD tear volumes were significantly greater compared with baseline (p = 0.028, Wilcoxon test). Histopathologic examination of the lacrimal glands showed no discernible tissue damage from chronic neurostimulation. In addition, there were no gross adverse effects on the general well-being of the animals due to chronic stimulation. LNS with an IPG appears to increase aqueous tear production. Chronic LNS showed no histopathologic lacrimal gland damage. This study suggests that LNS is a promising new treatment strategy to increase aqueous tear production.

Near-field discrimination of sound source distance in the rabbit.

The acoustical cues and physiological processing mechanisms underlying the perception of the distance of sound sources are not well understood. To understand the relation between physiology and behavior, a first step is to use an animal model to study distance sensitivity. The goal of these experiments was to establish the capacity of the Dutch-belted rabbit to discriminate between sound sources at two distances. Trains of noise bursts were presented from speakers that were located either directly in front of the rabbit or at a 45 ° angle in azimuth. The reference speaker was positioned at distances of 20, 40, and 60 cm from the subject, and the more distant test speaker was systematically moved to determine the smallest difference in distance that could be reliably discriminated by the subject. Noise stimuli had one of three bandwidths: wideband (0.1-10 kHz), low-pass (0.1-3 kHz), or high-pass (3-10 kHz). The mean stimulus level was 60 dB sound pressure level (SPL) at the location of the rabbit's head, and the level was roved over a 12-dB range from trial to trial to reduce the availability of level cues. An operant one-interval two-alternative non-forced choice task was used, with a blocked two-down-one-up tracking procedure to determine the distance discriminability. Rabbits were consistently able to discriminate two distances when they were sufficiently separated. Sensitivity was better when the reference distance was 60 cm at either azimuth (distance ratio = 1.5) and was worse when the reference distance was 20 cm (distance ratio = 2.4 at 0 ° and 1.75 at 45 °).

Silk hydrogels for sustained ocular delivery of anti-vascular endothelial growth factor (anti-VEGF) therapeutics

Silk hydrogels were formulated with anti-vascular endothelial growth factor (anti-VEGF) therapeutics for sustained ocular drug delivery. Using silk fibroin as a vehicle for delivery, bevacizumab-loaded hydrogel formulations demonstrated sustained release of 3months or greater in experiments in vitro as well as in vivo using an intravitreal injection model in Dutch-belted rabbits. Using both standard dose (1.25mg bevacizumab/50μL injection) and high dose (5.0mg bevacizumab/50μL injection) hydrogel formulations, release concentrations were achieved at day 90 that were equivalent or greater than those achieved at day 30 with the positive standard dose control (single injection (50μL) of 1.25mg bevacizumab solution), which is estimated to be the therapeutic threshold based on the current dosage administration schedule of 1 injection/month. These gels also demonstrated signs of biodegradation after 3months, suggesting that repeated injections may be possible (e.g., one injection every 3–6months or longer). Due to its pharmacokinetic and biodegradation profiles, this delivery system may be used to reduce the frequency of dosing for patients currently enduring treatment using bevacizumab or other anti-VEGF therapeutics.

Intravitreal administration of known phototoxicants in the rabbit fails to produce phototoxicity: implications for phototoxicity testing of intravitreally administered small molecule therapeutics

Context: Intravitreal (ITV) dosing has become a clinically important route of administration for the treatment of uveitis, endophthalmitis, retinal vein occlusion, diabetic macular edema and age-related macular degeneration. Despite this, there are no validated non-clinical models of phototoxicity for ITV products.Objective: The objective of this study was to develop an ITV rabbit model of phototoxicity for use in assessing the photosafety of small molecules therapeutics.Materials and methods: Dutch Belted rabbits were intravitreally injected bilaterally with four known phototoxicants: 8-methoxypsoralen, lomefloxacin, doxycycline and stannsoporfrin. Triescence®, a non-phototoxic triamcinolone acetonide steroid formulation designed for ITV administration, was used as a negative control. One eye was then irradiated with solar-simulated ultraviolet radiation for 30 min, 1 h after dosing, while the other eye was occluded, serving as a non-irradiated control.Results: Despite the direct administration of known phototoxicants into the vitreous, no evidence of ocular phototoxicity was observed in any dose group. Direct (non-phototoxic) retinal toxicity was observed in the doxycycline dose group only.Conclusion: These data suggest that the posterior segment of the rabbit eye is protected against phototoxicity by anatomical and/or physiological mechanisms, and is not a useful model for the assessment of phototoxicity of intravitreally administered molecules.

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167–384 nM; Emax = 32–86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g. IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60–84 nM; Emax = 29–44% relative to max. BK-induced effects). FR-190997 (0.3–300 μg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g. 34.5 ± 7.5% decrease; 6 h post-dose of 30 μg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.

Comparison of ocular tissue weights (volumes) and tissue collection techniques in commonly used preclinical animal species.

AbstractPurpose Ocular pharmacokinetic and distribution studies are key elements for the development of ophthalmic drugs and are used to correlate drug levels in preclinical species with human dosages and exposures. The purpose of this study was to compare ocular tissue weights for five commonly used preclinical species using two collection techniques.Methods Standard ocular tissues were collected from five males and five females of each species; New Zealand white (NZW) and Dutch Belted (DB) rabbits, beagle dogs, Gottingen minipigs, and Cynomolgus monkeys. Two collection techniques were used; a collection from fresh eyes (OS, immediately following enucleation) or a collection from frozen eyes (OD, following flash freezing). A specific procedure and collection sequence was followed for each technique.Results Aqueous and vitreous humor, cornea, lens, iris‐ciliary body, retina, choroid, and sclera were collected from each species; these tissues represented mean values of 70.0‐82.0% and 84.3‐88.9% of the total eye weight, respectively, for fresh and frozen collection. Tissue recovery levels were consistently higher using the frozen collection technique. Mean combined male &amp; female vitreous humor weights using the frozen collection were 1.41, 1.31, 2.22, 2.34, and 2.04 g for NZW &amp; DB rabbits, dogs, minipigs, and monkeys, respectively. Other tissue weights will be compared.Conclusion Comparative ocular tissue weights are presented for common preclinical species using fresh or frozen collection techniques. Vitreous humor and other tissue weights collected by the frozen technique accurately represent the weight (and volume assuming a VH density of 1) of the total tissue for comparative purposes to human vitreous volumes.

Toxicological and pharmacokinetic properties of QPI-1007, a chemically modified synthetic siRNA targeting caspase 2 mRNA, following intravitreal injection.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA (siRNA), QPI-1007, following intravitreal administration. QPI-1007 is a chemically modified siRNA designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the caspase 2 protein and is being developed as a neuroprotectant for the treatment of nonarteritic anterior ischemic optic neuropathy and other optic neuropathies such as glaucoma that result in the death of retinal ganglion cells. The half-life of QPI-1007 in the vitreous and retina/choroid in the Dutch Belted rabbit was about 2 days, and there was no sign of accumulation after repeated administrations at either 2- or 4-week dosing intervals in the rabbit. QPI-1007 was well tolerated in Dutch Belted rabbits following single or repeated intravitreal administrations of up to 11 doses over 9 months. Test-article-related effects were limited to the eyes, with minimal to mild vitreal cellular infiltration being the major finding, which was reversible. In repeated-dose studies, a modest reduction in B-wave amplitude obtained by electroretinography was observed in animals treated with the highest dose level tested (3 mg, which is equivalent to a 12 mg/eye human dose) that was not considered to be clinically meaningful. Administration in the rat of either a single bolus intravenous (i.v.) injection of 100 mg/kg or daily bolus i.v. injections of 75 mg/kg/day for 28 days failed to elicit any macroscopic or microscopic changes, suggesting a low risk for systemic toxicity. QPI-1007 was negative in three genetic toxicity studies. Overall, the nonclinical studies support the further development of QPI-1007.

SUBVISIBLE RETINAL LASER THERAPY

Laser therapy for diabetic macular edema and other retinal diseases has been used within a wide range of laser settings: from intense burns to nondamaging exposures. However, there has been no algorithm for laser dosimetry that could determine laser parameters yielding a predictable extent of tissue damage. This multimodal imaging and structural correlation study aimed to verify and calibrate a computational model-based titration algorithm for predictable laser dosimetry ranging from nondamaging to intense coagulative tissue effects. Endpoint Management, an algorithm based on a computational model of retinal photothermal damage, was used to set laser parameters for various levels of tissue effect. The algorithm adjusts both power and pulse duration to vary the expected level of thermal damage at different percentages of a reference titration energy dose. Experimental verification was conducted in Dutch Belted rabbits using a PASCAL Streamline 577 laser system. Titration was performed by adjusting laser power to produce a barely visible lesion at 20 ms pulse duration, which is defined as the nominal (100%) energy level. Tissue effects were then determined for energy levels of 170, 120, 100, 75, 50, and 30% of the nominal energy at 1 hour and 3, 7, 30, and 60 days after treatment. In vivo imaging included fundus autofluorescence, fluorescein angiography, and spectral-domain optical coherence tomography. Morphologic changes in tissue were analyzed using light microscopy, as well as scanning and transmission electron microscopy. One hundred and seventy percent and 120% levels corresponded to moderate and light burns, respectively, with damage to retinal pigment epithelium, photoreceptors, and at highest settings, to the inner retina. 50% to 75% lesions were typically subvisible ophthalmoscopically but detectable with fluorescein angiography and optical coherence tomography. Histology in these lesions demonstrated some selective damage to retinal pigment epithelium and photoreceptors. The 30% to 50% lesions were invisible with in vivo multimodal imaging, and damage was limited primarily to retinal pigment epithelium, visible best with scanning electron microscopy. Over time, photoreceptors shifted into the coagulated zone, reestablishing normal retinal anatomy in lesions ≤100%, as seen in optical coherence tomography and light microscopy. Transmission electron microscopy at 2 months demonstrated restoration of synapses between shifted-in photoreceptors and bipolar cells in these lesions. Retinal pigment epithelium monolayer restored its continuity after 1 week in all lesions. No damage could be seen <30% level. A retinal laser dosimetry protocol based on the Endpoint Management algorithm provides reproducible changes in retinal morphology in animals with various levels of pigmentation. This algorithm opens doors to clinical trials of well-defined subvisible and nondestructive regimes of retinal therapy, especially important for treatment of macular disorders.

Trabecular Meshwork Bradykinin Receptors: mRNA Levels, Immunohistochemical Visualization, Signaling Processes Pharmacology, and Linkage to IOP Reduction

To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. Previously documented procedures were utilized throughout these studies. Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-β2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 μg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 μg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

An Experimental Steroid Responsive Model of Ocular Inflammation in Rabbits Using an SLT Frequency Doubled Q Switched Nd:YAG Laser

To develop a minimally invasive rabbit model of postoperative anterior chamber (AC) inflammation using a commercially available frequency doubled Nd:YAG laser [intended for selective laser trabeculoplasty (SLT)]. Escalating laser energy was applied to the iris of male Dutch-belted rabbits and the subsequent inflammatory response was observed to determine the laser dose required to generate self-limiting inflammation of at least 3 days' duration. In subsequent experiments, 10 eyes of 10 male Dutch-belted rabbits underwent baseline slit lamp examination, intraocular pressure (IOP), and AC flare meter readings. Starting 1 day before laser application, 5 animals received topical 20 μL dexamethasone 1% to 1 eye 4 times daily for 5 days. Five control animals were treated with saline. Masked assessments of flare, cells, and IOP were made daily for 7 days. Histopathologic changes were assessed in enucleated eyes. Compared to controls, dexamethasone-treated rabbits had less postlaser AC flare on postoperative day (POD)2 (19±5 vs. 44±21photons/ms, P=0.03) and POD3 (16±9 vs. 33±11 photons/ms, P=0.03). In dexamethasone-treated rabbits, clinically graded flare (on POD1) and cells (on POD1 and 2) were lower than controls, but did not reach statistical significance. In the control group, IOP was significantly lower than the dexamethasone-treated group on POD2 (14.1±3.4 vs. 19.8±1.1 mmHg, P=0.03) and POD3 (14.2±2.2 vs. 19.0±2.2 mmHg, P=0.01). Histopathology showed pigment clumping and changes limited to anterior layers of the iris. Commercially available SLT laser can be used to create a minimally invasive, steroid-responsive animal model of anterior uveitis with the potential for use in the evaluation and comparison of drugs intended to treat AC inflammation.

Polychromatic Angiography for the Assessment of VEGF-Induced BRB Dysfunction in the Rabbit Retina

To determine the utility of polychromatic angiography (PCA) in the assessment of VEGF-induced blood retinal barrier (BRB) dysfunction in rabbits. Twenty-six eyes of 24 Dutch Belted rabbits were injected intravitreally with 1.25 μg (group A, n = 5), 10 μg (group C, n = 7), or 4 μg (group B, n = 6; group D, n = 4; and group E, n = 4) of VEGF on day 0. Groups D and E were also injected intravitreally with 1.25 μg and 12.5 μg bevacizumab, respectively, on day 2. On days 0, 2, 4, 7, 11, and 14, PCA was performed using a contrast agent mixture composed of fluorescein sodium, indocyanine green, PCM102, and PCM107 and imaged with a modified fundus camera. PCA scores were based on detected leaking fluorophores. On day 7, there was a statistically significant difference between PCA scores of group A (0.6 ± 0.89) and both groups B (2.67 ± 1.37, P = 0.0154) and C (3.33 ± 0.52, P = 0.00085). There was also a statistically significant difference between groups B and E (PCA score 0.75 ± 0.96, P = 0.032) on day 7. On day 11, there was statistically significant difference between group C (1.80 ± 1.1) and both groups A (0, P = 0.021) and B (0.33 ± 0.52, P = 0.037). A differential response to both increasing VEGF dose and administration of bevacizumab could be discerned using the PCA. PCA allowed stratification of VEGF-induced BRB dysfunction and inhibitory effects of bevacizumab therapy in the rabbit retina.

24h IOP control by subchronic AMA0076 administration in Dutch Belted rabbits

AbstractPurpose To investigate IOP lowering efficacy of rho kinase (ROCK) inhibitors, AMA0076 and Y‐39983, after subchronic treatment.Methods Dutch Belted rabbits (5 rabbits/concentration) were treated daily with a single topical dose of AMA0076 (0.05, 0.1 and 0.4%) or Y‐39983 (0.025, 0.05 and 0.1%) in a masked fashion for 14 days. Saline was used as control in the contralateral eye. IOP was measured before, 3 and 7 hours after administration of the treatment. AMA0076 is a locally acting ROCK inhibitor, to reduce side effects such as hyperemia otherwise seen after administration of a ROCK inhibitor.Results Mean baseline IOP before the start of the experiment was 21.02 ± 0.42 and 20.69 ± 0.39 mmHg for AMA0076 and Y‐39983, respectively. Significant IOP reduction was observed in all AMA0076 and Y‐39983 treatment groups. A maximal IOP reduction of 39 and 35% was reached with AMA0076 0.1% and Y‐39983 0.05%, respectively. Initially, IOP returned to baseline values 24h after administration of AMA0076. After 3 days of treatment with AMA0076 a sustained IOP lowering effect was present, IOP did not return to baseline values, but remained significantly lower for all concentrations of AMA0076 (p&lt;0.05). Treatment with Y‐39983 did not show this sustained IOP decrease. As a result of this sustained IOP lowering effect smaller IOP fluctuations were observed in animals treated with AMA0076 compared to Y‐39983. Hyperemia was observed with all concentrations of Y‐39983 whereas only mild hyperemia was induced after administration of AMA0076 0.4%.Conclusion Once daily treatment of rabbits with ROCK inhibitor AMA0076 resulted in IOP reduction that was more sustained and associated with smaller peak‐through fluctuations than Y‐39983.

Ocular and systemic distribution of MRZ‐99030 in pigmented mouse, rat, rabbit and non‐human primate by quantitative tissue analysis

AbstractPurpose The ocular tissue distribution and systemic pharmacokinetics of MRZ‐99030 was determined after single or repeated topical ocular dose regimens of MRZ‐99030 to pigmented mice, rats, rabbits, and non‐human primates. MRZ‐99030, an amyloid‐β aggregation modulator, is currently under development for the treatment of glaucoma and AMD, which are the leading causes of blindness and represent diseases with high unmet medical need.Methods Dutch Belted rabbits, Cynomolgus monkeys, Dark Agouti rats, and C57/BL6 mice received single or repeated bilateral topical ocular doses of MRZ‐99030. Ocular tissues and plasma were collected up to 24h after dosing and analyzed for MRZ‐99030 by liquid chromatography‐mass spectrometry (LC‐MS).Results MRZ‐99030 was readily absorbed into ocular tissues for all species with highest concentrations observed in the cornea and conjunctiva. MRZ‐99030 was also found in the ocular tissues of the posterior segment (choroid with RPE and retina) at all time points. Pharmacokinetics show MRZ‐99030 in all tissues and systemically, with elimination half‐lives that indicate somewhat sustained concentrations in melanin containing tissues. Concentrations in ocular tissues, in particular for the retina, increase with an increase in dose level (mouse and rat) and dose frequency (all species).Conclusion The results show that MRZ‐99030 is absorbed into the ocular tissues in all species following topical administration and is readily distributed to the posterior segment of the eye, including the retina. Therefore, this finding strongly supports that MRZ‐99030 is able to modulate the Aß aggregation in the target tissue of glaucoma and AMD.