Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

Glaucoma is a leading cause of world blindness, often associated with elevated eye pressure. Current glaucoma treatments aim to lower eye pressure by improving aqueous humor outflow from the eye. Ocular lymphatics have been demonstrated to contribute to aqueous humor outflow in human and sheep. It is not known whether any glaucoma drugs target this lymphatic drainage. The mouse is a valuable model with similar aqueous humor dynamics and pharmacology as human. Using in vivo hyperspectral fluorescence imaging combined with intracameral quantum dot injection, we identified an ocular lymphatic drainage in mouse. Immunofluorescence and confocal microscopy revealed lymphatic channels in the ciliary body, sclera, and orbit that may be responsible for this lymphatic drainage. We showed that latanoprost, a prostaglandin F2α analog widely used to treat glaucoma, increases this ocular lymphatic drainage. Our findings provide the framework for future development of novel glaucoma drugs that stimulate the ocular lymphatic drainage.

This project explores the use of a novel sustained release mucoadhesive micelle-based drug delivery system in combination with 0.005% latanoprost (LTP) on our partially open angle mouse model of glaucoma (AP-2β TMR-KO). We previously tested for LTP treatment in our model and found a reduction in intraocular pressure (IOP) 20 minutes following treatment. This information led us to investigate the long-term effect of LTP treatment and micelle loaded with LTP (MLTP) treatment in our model. We hypothesized that the MLTP treatment would be more effective in reducing IOP and preventing glaucomatous effects than LTP treatment alone in the AP-2β TMR-KO mice. The MLTP groups of animals (wildtype and mutant) were treated every 3 days, and this was compared with animals treated with LTP daily as well as animals treated every 3 days with LTP alone for comparison’s sake for 60 days. IOP measurements were taken every 3 days. Following long term LTP treatment alone, mutant mice showed a consistent decrease in their baseline IOPs with a significant reduction in baseline IOP at 35 days of treatment across all cohorts (P<0.0001). In comparison, mutants treated with MLTP exhibited an even greater reduction in baseline IOP following long term treatment. After the treatment period, mice were euthanized, and their eyes were enucleated, fixed, sectioned, and stained for retinal ganglion cells (RGCs) using Brn3a. Mutant mice exhibited a significant decrease in RGC cell number when compared to wildtype, and this loss was not rescued by treatment with LTP. However, mutants treated with MLTP demonstrated significant RGC cell protection compared to eyes of untreated mutants, as well as everyday LTP treated mutants. / Thesis / Master of Science (MSc) / An effective treatment strategy is required to prevent irreversible blindness caused from glaucoma. Unfortunately, compliance with current medications is extremely poor, as they require frequent administration due to their low ocular bioavailability and short-term effect. As such, this thesis aims to explore an alternative drug delivery approach in a partially open angle mouse model of glaucoma to prevent the worsening of glaucoma and ultimately improve patient compliance.

The discovery of the ocular hypotensive effect of topically applied prostaglandins (PGs) has raised a number of questions about the mechanisms of action involved. The aim of the present thesis was to answer some of these questions. PGs reduce the intraocular pressure (IOP) by increasing uveoscleral flow through the ciliary muscle, but the exact mechanism is not known. Morphological changes may be involved. PGs are also involved in the inflammatory response. In the first study the aim was to investigate the effect of latanoprost, a prostaglandin F2 a-analogue, on the blood-aqueous barrier and the IOP restoration after long-term treatment. 26 glaucoma patients were treated with latanoprost (50 pg/ml) once daily for 6-12 months. Aqueous protein concentration was followed with a laser flare meter in 16 patients throughout this period. No change was observed. IOP increased slowly after withdrawal of treatment. It was concluded that latanoprost has no clinically significant effect on the permeability of the blood-aqueous barrier and that the IOP will return to pretreatment levels within a few weeks, indicating that any changes in the ciliary muscle morphology are reversible. In 20 healthy volunteers it was attempted to prevent the ocular hypotensive effect of latanoprost by inhibiting uveoscleral flow by a pronounced ciliary muscle contraction. For this purpose a high dose of the cholinergic agonist, physostigmine (1 drop 8 mg/ml alternate hours) was used. However, the effects on IOP of the two drugs were mainly additive most likely due to a short-lasting effect of physostigmine on the ciliary muscle. The progressive IOP reduction by physostigmine in the second study raised the question as to whether the drug reduces aqueous flow apart from enhancing outflow. On the contrary, in the third study repeated administrations of physostigmine, in 20 normal subjects, increased aqueous flow, measured with fluorophotometry, by about 25%. From studies of patients it is known that latanoprost twice daily has less ocular hypotensive effect than once daily. This was the subject of the two remaining studies. The possibility that latanoprost causes a short-lasting increase in aqueous flow was examined in 18 healthy volunteers. Application of a second drop in the morning would blunt some of the early IOP lowering effect of latanoprost. Once or twice daily applications had similar effect on aqueous flow, a tendency to an increase without any difference between the dose regimens. The next study confirmed the difference in effect on IOP between once and twice daily applications in 40 normal subjects. The difference remained even when one of the two applications was omitted after two weeks’ treatment. The results indicate that applying latanoprost twice daily induces a modest receptor desensitisation. / <p>Diss. Umeå : Umeå universitet, 1997, härtill 5 uppsatser.</p> / digitalisering@umu

This thesis investigates the feasibility of loading silicone hydrogel (SiHy) contact lenses with two different hydrophobic therapeutics, latanoprost and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), for treatment of glaucoma and hyperemia respectively. The two methods of loading were 1) thermal loading in an aqueous medium and 2) convective loading in a solution of n-propanol. Dailies Total1® lenses prepared in this manner were tested for their loading and their release into artificial tears. Continuous release over 1-4 days at therapeutic levels is achievable from thermal loading of DMPC, convective loading of DMPC, and convective loading of latanoprost. The DMPC loading processes can be naturally integrated into standard manufacturing lines for Dailies Total1®. Both DMPC and latanoprost release at rates proportional to the amount loaded into a contact lens. Latanoprost loads into a contact lens strictly proportionally to the loading concentration and the time of loading. The convective loading step represents a significant improvement on both the time of loading (reduced from days to minutes) and the loading capacity of silicone hydrogel contact lenses. This thesis also compares the loading and release of latanoprost in the convective loading procedure using the SiHy contact lenses of Acuvue Advance® (Johnson & Johnson Vision Care, Jacksonville, FL) , Air Optix® (Alcon, Copenhagen, Denmark), Biofinity® (CooperVision), PureVision® (Bausch & Lomb), and Dailies Total1® (Alcon), and the polyHEMA lens, SofLens 38® (Bausch & Lomb), finding that silicone hydrogels load an order of magnitude more drug than the polyHEMA lens and release into artificial tears for an order of magnitude longer. Overall, these experiments provide a quantitative understanding of the dynamics of loading and release for both DMPC and latanoprost.

<p>Two different principles were studied. 1st - statistical analysis techniques were used to obtain medical results from a patient population. 2nd - the patient population was used to study the statistical analysis techniques. </p><p>Medical conclusions: latanoprost and timolol treatment showed a statistically significant and clinically useful mean IOP-reduction in a typical worldwide clinical trial population. Latanoprost reduced the IOP 1.6 mm Hg more than timolol. The IOP-reduction was maintained with timolol and slightly enforced with latanoprost up to 6 months of treatment. The mean IOP-reduction was maintained during 2 years of latanoprost treatment. The overall risk of withdrawal due to insufficient IOP-reduction with latanoprost was 8%. </p><p>The statistical methodological issues are of a general and reoccurring character in trial design of the IOP-reduction: should the statistical hypothesis testing be based on the mean intraocular pressure (IOP) or the proportion of patients who reach a specific IOP level, should the estimate of the IOP or IOP-reduction be based on single eyes, mean of bilaterally eligible and identically treated eyes or the difference between an eye with active treatment and a placebo treated contralateral eye, and is mean of replicated recordings useful? Statistical methodological conclusions: the most effective response variable varies with the selected patient population. Therefore, the trial design process should include a comparison of the variability, test power and required sample size for the possible response variables in a sample of the target population. At minimum a statistical consideration should be done.</p>

Two different principles were studied. 1st - statistical analysis techniques were used to obtain medical results from a patient population. 2nd - the patient population was used to study the statistical analysis techniques. Medical conclusions: latanoprost and timolol treatment showed a statistically significant and clinically useful mean IOP-reduction in a typical worldwide clinical trial population. Latanoprost reduced the IOP 1.6 mm Hg more than timolol. The IOP-reduction was maintained with timolol and slightly enforced with latanoprost up to 6 months of treatment. The mean IOP-reduction was maintained during 2 years of latanoprost treatment. The overall risk of withdrawal due to insufficient IOP-reduction with latanoprost was 8%. The statistical methodological issues are of a general and reoccurring character in trial design of the IOP-reduction: should the statistical hypothesis testing be based on the mean intraocular pressure (IOP) or the proportion of patients who reach a specific IOP level, should the estimate of the IOP or IOP-reduction be based on single eyes, mean of bilaterally eligible and identically treated eyes or the difference between an eye with active treatment and a placebo treated contralateral eye, and is mean of replicated recordings useful? Statistical methodological conclusions: the most effective response variable varies with the selected patient population. Therefore, the trial design process should include a comparison of the variability, test power and required sample size for the possible response variables in a sample of the target population. At minimum a statistical consideration should be done.