Roy Chowdhury, Uttio

AbstractGlaucoma, a progressive neurodegenerative ocular disease, is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is the most common—and the only treatable—risk factor for glaucoma. IOP is generated by the balance between production and removal of aqueous humor in the anterior part of the eye, and the latter happens either through the uveoscleral or the conventional pathway. Although both pathways are important for aqueous humor removal, dysfunction within the conventional pathway is more commonly associated with increased resistance leading to elevated IOP and glaucoma. The conventional pathway can be separated into proximal (trabecular meshwork and inner wall of Schlemm's canal) and distal (outer wall of Schlemm's canal, collector channels, and episcleral vasculature) regions. Both regions contribute resistance to aqueous humor removal, but the proximal region has been studied more extensively due to the availability of model systems. In contrast, little is known about the role of the distal region in outflow resistance, largely due to the lack of suitable in vitro models. To address this, we have developed a novel method of isolating and culturing vascular distal outflow pathway (VDOP) cells from the distal outflow region of human eyes. VDOP cells can be used to study the physiological and molecular functions of cells in the distal outflow region and can help in the development of ocular hypotensive drugs that specifically target this area. We also provide a protocol describing immunohistochemical methods to validate the molecular profile of these cells, utilizing cell surface markers that distinguish them from adjacent cells. © 2022 Wiley Periodicals LLC.Basic Protocol 1: Isolation and culture of VDOP cellsBasic Protocol 2: Analysis of cell surface markers

Purpose: To evaluate pharmacokinetic parameters and ocular hypotensive effects of cromakalim prodrug 1 (CKLP1) in normotensive large animal models. Methods: Optimal CKLP1 concentration was determined by dose response and utilized in short- (5–8 days) and long-term (60 days) evaluation in hound dogs ( n = 5) and African Green Monkeys ( n = 5). Blood pressure was recorded 3–5 times per week with a tail cuff. Concentrations of CKLP1 and the parent compound levcromakalim were assessed in hound dog plasma and select tissues by LC-MS/MS after bilateral ocular treatment with CKLP1 for 8 days. Pharmacokinetic parameters were calculated from days 1, 4, and 8 data. After necropsy, histology was assessed in 43 tissue samples from each animal. Results: In hound dogs and African Green monkeys, 10 mM CKLP1 (optimal concentration) significantly lowered intraocular pressure (IOP) by 18.9% ± 1.1% and 16.7% ± 6.7%, respectively, compared with control eyes ( P < 0.05). During treatment, no significant change in systolic or diastolic blood pressure was observed in either species ( P > 0.1). Average values for half-life of CKLP1 was 295.3 ± 140.4 min, C max, 10.5 ± 1.6 ng/mL, and area under the concentration vs. time curve (AUC last ) 5261.4 ± 918.9 ng·min/mL. For levcromakalim, average values of half-life were 96.2 ± 27 min, C max 1.2 ± 0.2 ng/mL, and AUC last 281.2 ± 110.8 ng·min/mL. No significant pathology was identified. Conclusions: CKLP1 lowered IOP in hound dogs and African green monkeys with no effect on systemic blood pressure. Ocular topical treatment of CKLP1 showed excellent tolerability even after extended treatment periods.