CFTR
act-K267 was quantified by liquid chromatography/mass spectroscopy (LC/MS) at 15 minutes, 1 hour, 2 hours, 6 hours, and 24 hours after a single, 3-nmol topical dose. To recover CFTR
act-K267 at indicated times, three eye washes of 30 μL sterile PBS were done with solution recovered from the lateral and medial canthi after manual eyelid blinking using 50-μL glass capillary tubes. Pooled washes were diluted in three volumes of ethyl acetate, centrifuged at 18,500
g for 15 min, and the collected supernatant was analyzed by LC/MS (Waters 2695 HPLC with Micromass ZQ).
16 LC was done on an Xterra MS C18 column (2.1 mm × 100 mm, 3.5 μm) with 0.2 mL/min water/acetonitrile containing 0.1% formic acid, 12-minute linear gradient, and 5% to 95% acetonitrile.
In chronic treatment studies, CFTRact-K267 (3 nmol, or formulation control) was given twice daily (8 AM and 4 PM) for 28 days. Two mL of blood was collected from the marginal ear vein in EDTA tubes. Rabbits were then euthanized using 150 mg/kg euthasol, and ocular tissues, blood, and peripheral organs were collected. Using a surgical microscope, 150 μL of aqueous humor was collected through the peripheral cornea using a 25-gauge needle, and 300 μL of vitreous fluid was aspirated through the pars plana with a 23-gauge needle. Following transcardial perfusion with heparinized PBS, the eyes were enucleated with lids intact, and the cornea, iris/ciliary body, lens, bulbar, forniceal and palpebral conjunctiva, and retina of both eyes were dissected, weighed, homogenized in a 1:4 mixture of water:ethyl acetate (10 mL/1 g tissue), and centrifuged (1000g for 15 minutes). Plasma, aqueous, and vitreous samples were each mixed with 3 volumes of ethyl acetate and centrifuged for 15 minutes at 18,500g, and the supernatant was evaporated and redissolved in HPLC eluent (100 μL of 1:3 water:acetonitrile containing 0.1% formic acid) for LC/MS analysis. Also, the brain, kidney, heart, and liver were removed, weighed, mixed in a 1:4 mixture of water:ethyl acetate (10 mL/1g tissue), and homogenized. The homogenized samples were vortexed and centrifuged (1000g for 15 minutes), and the ethyl acetate-containing supernatant was evaporated and then redissolved in HPLC eluent for LC/MS analysis. The lower limit of detection for CFTRact-K267 was ∼0.2 pg/mg of homogenized tissue or biological fluid, which was defined as giving a signal-to-noise ratio >3.