Evaluation of delivery agents used for introduction of small interfering RNAs into feline corneal cells.

OBJECTIVE: To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)-specific siRNA combinations. SAMPLE: Feline primary corneal cells and 19 six-month-old colony-bred cats. PROCEDURES: siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells. RESULTS: Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1-specific mRNA, 63% to 67% reduction of FHV-1-specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo. CONCLUSIONS AND CLINICAL RELEVANCE: The tested anti-FHV-1-specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.

Effects of oral administration of methazolamide on intraocular pressure and aqueous humor flow rate in clinically normal dogs.

OBJECTIVE: To determine magnitude and duration of the effect of oral administration of methazolamide at 2 dosages on intraocular pressure (IOP) in dogs in single-dose and multiple-dose trials and to determine aqueous humor flow rate (AHFR) by use of anterior segment fluorophotometry before and during treatment. ANIMALS: 25 healthy adult Beagles. PROCEDURE: Baseline IOPs and AHFRs were determined on days 0 and 1, respectively. On day 2, the single-dose trial was initiated with oral administration of 25 or 50 mg of methazolamide at 7 AM to 2 groups of 10 dogs each. Five dogs served as controls. In the multiple-dose trial, the same dogs received 25 or 50 mg of methazolamide at 7 AM and at 3 and 11 PM on days 3 through 9. RESULTS: Intraocular pressures varied diurnally with highest IOPs in the morning. In the single-dose trial, IOP decreased significantly at 3 to 6 hours after treatment and then increased significantly at later time points, compared with baseline values. In the multiple-dose trial, dogs in both treatment groups had significantly lower IOPs during the treatment period at 10 AM and 1 PM but not at 6 and 9 PM, compared with baseline values. In both treatment groups morning IOPs had returned to baseline values by the first day after treatment. Evening IOPs were significantly increased by 2 to 3 days after treatment, compared with baseline values. The AHFRs in both treatment groups were significantly lower than pretreatment AHFRs. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of methazolamide decreases IOPs and AHFRs in clinically normal dogs, with effectiveness diminishing in the evening.