Edward O. MacKay

Drug Delivery by Contact Lens in Spontaneously Glaucomatous Dogs

Purpose: The efficacy of ophthalmic drug delivery through contact lenses in animal model was explored to evaluate its potential for serving as an alternative to eye drops, which are inefficient vehicles for delivering ophthalmic drugs. Methods: The efficacy of timolol delivered via contact lenses was compared to eye drops in beagle dogs that suffer from spontaneous glaucoma. Experiments were conducted with NIGHT & DAY™ silicone hydrogel contact lenses and NIGHT & DAY™ loaded with vitamin E, which was included in the lens to extend the release duration of the drug. Timolol was loaded into contact lenses by soaking in drug/phosphate buffered saline solution, and the drug-loaded lenses were subsequently inserted in one of the eyes, with the other eye serving as control. The lenses were replaced every 24 hours, and the pharmacodynamics of intraocular pressure (IOP) and pupil size were monitored in both eyes. Results: The IOP reduction from baseline by NIGHT & DAY™ (5.02 ± 0.83 mmHg) was comparable with that by eye drops with similar drug dosing (4.64 ± 0.41 mmHg). In addition, lenses with one-third of the drug loading as eye drops resulted in the similar IOP reduction, suggesting higher bioavailability for contact lenses compared to eye drops. Inclusion of vitamin E into the lenses did not improve the IOP reduction. The IOP in the untreated eye also decreased from baseline for eye drops (3.17 ± 0.42 mmHg) but it remained relatively unchanged with treatments based on lenses, suggesting reduction in systemic absorption for delivery of drugs by contact lenses. Conclusions: Ophthalmic drug delivery through contact lenses increases bioavailability and reduces systemic drug uptake.

Mapping of the Disease Locus and Identification of ADAMTS10 As a Candidate Gene in a Canine Model of Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies.

Effect of different dose schedules of travoprost on intraocular pressure and pupil size in the glaucomatous Beagle

OBJECTIVE To evaluate changes in intraocular pressure and pupil size in glaucomatous dogs after instillation of 0.004% travoprost once in the morning, or once in the evening, or twice daily in 5-day multiple dose studies. MATERIALS AND METHODS Applanation tonometry (IOP) and pupil size (PS) measurements were obtained at 8 a.m., 10 a.m., 12 noon, 2 p.m. and 4 p.m. in eight glaucoma dogs. Methylcellulose (0.5% as placebo) was instilled in the control eye, and 0.004% travoprost was instilled in the opposite drug eye. Methylcellulose (0.5%) and 0.004% travoprost were instilled on the 2nd through to the 5th day with instillations in the morning (8.30 a.m.), or evening (8 p.m.), or twice daily (8.30 a.m. and 8 p.m.). RESULTS The mean +/- SEM diurnal changes from baseline IOP in the control and placebo eyes in all three studies ranged from 1.2 +/- 0.3 mmHg to 3.2 +/- 0.9 mmHg. The mean +/- SEM diurnal changes from the baseline IOP after 0.004% travoprost at 8 a.m. once daily for the next 4 days were 19.0 +/- 2.7 mmHg, 24.7 +/- 2.7 mmHg, 24.9 +/- 3.1 mmHg, and 24.7 +/- 3.1 mmHg, respectively, and were significantly different from the control eye. After travoprost was instilled at 8 p.m., the mean +/- SEM baseline changes from the baseline IOP in the drug eyes were 23.5 +/- 2.2 mmHg, 24.2 +/- 2.2 mmHg, 24.5 +/- 2.3 mmHg, and 24.2 +/- 2.3 mmHg, respectively. When 0.004% travoprost was instilled twice daily, the mean +/- SEM baseline IOP changes were 27.7 +/- 2.1 mmHg, 28.1 +/- 2.1 mmHg, 28.4 +/- 2.2 mmHg, and 28.5 +/- 2.2 mmHg, respectively, and were significantly different from the control eyes. Miosis of varying duration was frequent during the three studies. CONCLUSION Travoprost instilled once daily (a.m. or p.m.) as well as twice daily produces significant decreases in IOP and PS in the glaucomatous Beagle.

Histomorphometry of the optic nerves of normal dogs and dogs with hereditary glaucoma.

The beagle dog with hereditary primary open-angle glaucoma, unlike other animal models of human glaucoma, possesses a slowly progressive, sustained elevation of intraocular pressure. The effects of this insidious elevation in intraocular pressure on the axons of the optic nerves of three beagles at early stages of glaucoma and two beagles with advanced signs of glaucoma were compared to the optic nerves of four age-matched normal dogs. Plastic embedded optic nerve cross-sections (1 micron) 1 mm posterior to the lamina cribrosa were osmicated and stained with Toluidine Blue. Axons from 0.2 to > 2.0 microns in diameter were counted and measured in 16 cross-sectional regions of equal size within the whole optic nerve using a computerized image analysis system. The mean optic nerve axon diameters in the normal, early glaucomatous, and advanced glaucomatous dogs were 1.53, 1.25 and 1.13 microns respectively. The average total optic nerve axon count in the normal dogs was 148,303. Approximately 16% of the total axonal fibers were counted in each nerve. The counts of optic nerve axons 2.0 microns or greater in diameter were reduced by up to 60% in the central regions of the optic nerves of affected beagles. The large diameter axons of the peripheral optic nerve of the beagle dogs with glaucoma were more resistant to the elevated intraocular pressure. The counts of axons > 0.6 to 0.8 micron in diameter were significantly increased in glaucomatous beagles.

Effect of different dose schedules of bimatoprost on intraocular pressure and pupil size in the glaucomatous Beagle.

The changes in intraocular pressure and pupil size in glaucomatous dogs were evaluated after instillations of 0.03% bimatoprost (Lumigan, Allergan, Irvine, CA USA) once in the morning, or once in the evening, or twice daily in five day multiple dose studies. Applanation tonometry (IOP) and pupil size (PS) measurements were obtained at 8 am, 10 am, 12 noon, 2 pm, and 4 pm in 8 glaucoma dogs. Methylcellulose (0.5% as placebo) was instilled in the control eye, and 0.03% bimatoprost was instilled in the opposite drug eye. Methylcellulose (0.5%) and 0.03% bimatoprost were instilled the second through the fifth days with instillations in the morning (8:30 am), or evening (8 pm), or twice daily (8:30 am and 8 pm). The mean +/- SEM diurnal changes in IOP from baseline values after 0.03% bimatoprost at 8 am once daily for the next four days were 25.0 +/- 3.2 mm Hg, 25.6 +/- 2.9 mm Hg, 25.5 +/- 3.0 mm Hg, and 26.0 +/- 3.2 mm Hg respectively, and were significantly different from the control eye. After bimatoprost was instilled at 8 pm, the mean +/- SEM changes in IOP from baseline values in the drug eyes were 27.3 +/- 2.4 mm Hg, 26.6 +/- 2.2 mm Hg, 27.2 +/- 2.5 mm Hg, and 27.3 +/- 2.6 mm Hg respectively. When 0.03% bimatoprost was instilled twice daily, the mean +/- SEM changes in IOP from baseline values were 39.1 +/- 2.3 mm Hg, 39.9 +/- 2.2 mm Hg, 39.9 +/- 2.3 mm Hg, and 39.6 +/- 2.1 mm Hg respectively, and were significantly different from the control eyes. Miosis of varying duration was frequent during the three studies. Bimatoprost instilled once daily (am or pm) as well as twice daily produces significant decreases in IOP and PS in the glaucomatous Beagle.

Aqueous humor myocilin protein levels in normal, genetic carriers, and glaucoma Beagles

OBJECTIVE The gene (myocilin: MYOC) has been attributed to be involved in over 6% of inherited types of human glaucoma, the highest correlation for any gene to date. This study determines myocilin protein levels in the aqueous humor (AH) of normal laboratory quality, genetic carrier (offspring of normal laboratory quality and POAG Beagles), and primary open angle glaucoma (POAG) Beagles. MATERIALS AND METHODS Eighteen dogs were used and classified as either normal, carrier or having mild, moderate or advanced POAG. A 0.1 mL sample of AH was drawn from the anterior chamber of each dog in the study and frozen on dry ice. A modified Coomassie stain and Western blot, using a polyclonal rabbit antihuman myocilin antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), was run on each sample to compare the myocilin levels. A purified human trabecular meshwork excreted myocilin protein sample was used as a control (Alcon Research Laboratories, Fort Worth, TX) and its band/densitometry measurement was defined as one unit of myocilin for comparisons. RESULTS Comparisons of AH myocilin levels differed among normal laboratory quality, genetic carrier, and POAG Beagles at different stages of the disease. In the normal laboratory, Beagles the AH myocilin measured 0.817 +/- 0.075 units (mean +/- SEM); in the carrier Beagles the AH myocilin was 3.117 +/- 0.290 units. As POAG progressed, myocilin protein levels also increased to 6.097 +/- 0.810, 8.844 +/- 1.079, and 17.228 +/- 1.198 units in the early, moderate, and advanced forms, respectively. Overall comparisons between normal, carrier and all POAG Beagles combined showed significant differences (P < 0.0010). Individual comparisons between normal and carrier eyes showed significant differences (P < 0.0193). Comparisons between normal and all POAG eyes also showed significant differences (P < 0.0426). CONCLUSION This study shows myocilin protein is present in normal Beagles, markedly increased in POAG Beagles, and mildly increased in genetic carrier Beagles. There is a strong correlation between amounts of AH myocilin protein and the presence and severity of POAG. The exact role of AH myocilin levels in the genesis of ocular hypertension remains unresolved, but myocilin may adversely affect AH outflow.

Myocilin protein levels in the aqueous humor of the glaucomas in selected canine breeds

OBJECTIVE To compare aqueous humor myocilin protein levels in dogs with the primary glaucomas to those with the secondary glaucomas, primary cataracts, and diabetic cataracts. MATERIALS AND METHODS Four groups were selected, based on diagnosis by the attending veterinary ophthalmologists and included: primary glaucoma (primary open-angle glaucoma (POAG) and primary closed angle glaucoma (PCAG); n = 155); secondary glaucoma (n = 94); primary (presumed inherited) cataract (n = 142), and diabetic cataract (n = 83). A total of 474 samples (187 males, 263 females, 24 unreported) with average ages of 117 months for the males and 101 months for the females were analyzed. Myocilin protein was measured using the Coomassie staining and Western blot methods relative to a myocilin control. RESULTS Differences were seen between nonglaucomatous (cataractous) and glaucomatous dogs with myocilin levels in glaucomatous eyes being many times higher than those in the cataractous dogs. Primary glaucomatous dogs were found to have an aqueous humor myocilin protein level of 17.30 +/- 1.03 units. Secondary glaucomas had the highest level of myocilin in the aqueous humor with 19.27 +/- 1.41 units. Diabetic cataractous dogs had the lowest levels of myocilin reported with 6.60 +/- 0.88 (mean +/- SEM) units. Normal (cataractous) dogs had a myocilin level in the aqueous humor of 8.05 +/- 0.86 units. CONCLUSION Aqueous humor protein levels were elevated, relative to the myocilin control, in both the primary and secondary glaucoma groups compared to the cataract and diabetic cataract groups. Like in the Beagle POAG, aqueous humor myocilin protein levels are increased. Further studies are indicated to investigate the exact role of the aqueous humor myocilin protein in the genesis in increased IOP in these primary glaucomatous breeds.

Ocular parameters in a captive colony of fruit bats

OBJECTIVES To establish normal reference ranges of ocular parameters including phenol read thread, palpebral fissure length, horizontal and vertical corneal diameter, upright and hanging intraocular pressure (IOP) and to report ophthalmic examination findings of the anterior segment and lens, in a population of captive fruit bats. ANIMALS STUDIED Eyes of 30 bats of three species were included in this study: 10 (5 males, 5 females) Malayan Flying Foxes (Pteropus vampyrus), 10 (5 males, 5 females) Little Golden-mantled Flying Foxes (Pteropus pumilus), and 10 (4 males, 6 females) Island Flying Foxes (Pteropus hypomelanus). RESULTS The most common ophthalmic examination findings included iris-iris persistent pupillary membranes (83%), nuclear sclerosis (56.7%), prominent arterial circle (40%), iridal hyperpigmented foci (30%), pupillary margin cysts (27%), and third eyelid defects (20%). The mean, among all species for: phenol red thread was 20.23 ± 1.28 mm/15 s both eyes (OU); palpebral fissure length was 13.34 ± 0.33 mm for OU; for horizontal corneal diameter was 10.72 ± 0.32 mm for OU; for vertical corneal diameter was 9.90 ± 0.30 mm for OU; for the hanging intraocular pressures was 19.38 ± 0.77 mmHg for OU; for upright IOP was 13.95 ± 0.60 mmHg for OU. Measurements for the individual species groups and eyes were also calculated. CONCLUSIONS Results revealed the IOP of bats in a hanging position were significantly higher than the IOP of bats in an upright position. The size of the bat, between the species, affected palpebral fissure length, horizontal corneal diameter, and vertical corneal diameter. Information about the ocular structures and normal ophthalmic parameters for the Pteropus species is crucial for species protection because of dependence on vision for survival.

Dose response for travoprost® in the glaucomatous beagle.

OBJECTIVE To evaluate the changes in intraocular pressure (IOP) and pupil size in 12 Beagles with inherited glaucoma after instillations of 0.033, 0.0033, 0.001, 0.00033, and 0.0001% travoprost (Travatan®-Alcon Laboratories, Inc., Ft Worth, TX, USA) in multiple single-dose studies. PROCEDURES Intraocular pressure and pupil diameter (PD) measurements were obtained at 9 am, 12 pm, 3 pm, and 9 am the following day (24 h) in two groups of six glaucoma dogs. After 7 days, the vehicle or concentration was repeated in the contralateral eye of the same animals. RESULTS Concentrations of 0.00033, 0.001, and 0.0033% travoprost significantly lowered IOP and PD, but the 0.0001% concentration provided limited IOP changes, although PD changes were still significant. This suggests travoprost is effective in the dog to lower IOP and reduce pupil size at concentrations starting between 0.0001 and 0.00033%. CONCLUSIONS The dose response for travoprost in the glaucomatous Beagle indicates this model is highly sensitive to this group of drugs, even at concentrations as low as 0.00033% (1/12 the commercially available concentration).

Effect of Coherin™ on intraocular pressure, pupil size and heart rate in the glaucomatous Beagle: a pilot study

OBJECTIVE To evaluate effects of Coherin™ on intraocular pressure (IOP), pupil size (PS), and heart rate (HR) in glaucomatous Beagles in single-dose studies in a pilot study. MATERIALS AND METHODS Intraocular pressure, PS, and HR were measured in eight glaucomatous Beagles. One randomly chosen eye received single 50 μL doses of differing concentrations of Coherin™ (treated eye) or vehicle (placebo-treated eye), and the fellow eye served as the untreated control. After the first measurements, a single dose of either Coherin™ or sterile water vehicle was instilled in the drug and placebo eyes, respectively. RESULTS The mean ± SEM diurnal changes in IOP after 0.005%, 0.01%, 0.2%, 0.284%, 1%, 2%, and 4% topical Coherin™ once daily were 7.6 ± 3.2 mmHg, 15.5 ± 5.3 mmHg, 11.2 ± 4.4 mmHg, 11.8 ± 4.4 mmHg, 19.1 ± 3.8 mmHg, 5.0 ± 1.8 mmHg, and 8.8 ± 2.8 mmHg, respectively. The declines in IOP were significantly different (P < 0.05) from the untreated control eyes with the 0.2% and 0.284% Coherin™-treated eyes and suggestive for 1% Coherin™ concentrations. No signs of irritation, significant PS, and HR changes were detected in the Coherin™-treated eyes. CONCLUSION Of seven different concentrations, 2% and 0.248% Coherin™ produced significant declines in IOP in the glaucomatous beagle in single-dose studies when compared to both untreated control and placebo-treated eyes. One percent Coherin™ solution produced significant IOP decreases compared with the placebo-treated eye but not the untreated control eyes. No local ocular irritation, PS and HR changes were observed in Coherin™-treated eyes. This pilot study suggests that topical Coherin™ has potential as an ocular hypotensive agent.