Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease

Abstract

Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

Keywords: chloride channels; conjunctiva; cornea; ocular surface.

Figure 1.

Strategy for preclinical development of CFTR activators for dry eye therapy. Human WT CFTR activators identified by high-throughput screening are confirmed and characterized by electrophysiological and biochemical assays and then tested in live mice for activity at the ocular surface by measurements of potential difference and tear fluid volume. The best compounds are then tested for pharmacokinetic properties and efficacy in a dry eye mouse model.

Figure 2.

In vitro characterization of CFTR activators. A) Top: chemical structures. Bottom: representative I_sc measured in FRT cells expressing WT CFTR. CFTR current was stimulated by test compounds and FSK and inhibited by CFTR_inh-172 (10 μM). B) Concentration-dependence of CFTR activators. Each data set derived from a single dose–response experiment as in (A) and fitted to an exponential curve. One-hundred percent CFTR activation is defined as that produced by 20 μM FSK. C) I_sc measurement for VX-770, as in (A). D) Cellular cAMP concentration in FRT cells in response to incubation for 10 min with 5 µM test compounds without or with FSK (100 nM). Positive controls included FSK (100 nM and 20 μM) and FSK+IBMX (100 μM) (n = 4–8). Means ± sem.

Figure 3.

PD measurements of CFTR activators at the ocular surface in live mice. A) Left: photograph of an anesthetized mouse demonstrating ocular surface perfusion for PD measurement. The perfusion catheter, attached to the measuring electrode, is oriented perpendicular to the ocular surface. Cross-clamping forceps retract the upper eyelid to expose cornea and bulbar/palpebral conjunctiva for perfusion. The reference electrode is grounded via subcutaneous butterfly needle. Right: Idealized PD tracing for a typical experiment testing CFTR activity. B) Representative ocular surface PD measurements in WT mice. (Solution compositions are detailed in ref. 11). Concentrations: amiloride, 100 μM; FSK and CFTR_inh-172, 10 μM; test compounds, 1–10 μM, as indicated. C) Study as in B, but with VX-770, 1–10 μM, as indicated. D) Summary of ΔPD in WT mice produced by FSK (20 μM), or test compounds or VX-770 (each 1 μM). PDs were recorded in the presence of 100 μM amiloride and an outward apical Cl⁻ gradient. Means ± sem (n = 8–20 eyes per agonist tested). E) Representative ocular surface PD measurements in CF mouse. Study as in (B, C) CFTR_act-K032 (1–10 μM, as indicated).

Figure 4.

Tear fluid volume measurement of CFTR activators in living mice. A) Tear volume was measured just before and at the indicated times after single-dose topical application of vehicle (PBS, 0.5% polysorbate, 0.5% DMSO), cholera toxin (0.1 μg/ml), FSK (20 μM), or FSK+IBMX (250 μM). The effect of cholera toxin was measured after preanesthetizing the ocular surface with 4% lidocaine to suppress irritation and reflex tear secretion (n = 6–10 eyes per condition). Means ± sem. B) Time course of tear volume after topical delivery of the indicated compound. Concentrations: CFTR_act-B074, 100 μM; CFTR_act-J027, 50 μM; CFTR_act-K089, 50 μM; and VX-770, 10 μM (n = 6–18 eyes). Mean ± sem. C) Effect of repeated application. CFTR_act-J027 (0.1 nmol) was topically applied 3 times/d for 2 d. Tear volume measurements were obtained after doses 1 and 2 on d 1 and dose 5 on d 2 (n = 6 eyes). Means ± sem. D) Lack of effect of CFTR activators on tear volume in CF mice, with compounds tested at the same concentrations as in (B).

Figure 5.

Compound pharmacology. A) LC/MS determination of CFTR_act-K089 amount in tear fluid at indicated times after single-dose (0.1 nmol) administration. Representative background-subtracted peak areas from tear washes (left) and corresponding amounts recovered (right) (n = 4 eyes/time point). Means ± sem. Dashed lines: highest and lowest calculated quantities of CFTR_act-K089 necessary to achieve EC₅₀ concentration. B) Lissamine green staining of corneas in BALB/c mice, measured on a 12-point scale after 14 d of 3 times/d treatment with CFTR activators (0.1 nmol) or vehicle (n = 6 eyes per group). Means ± sem. Shown as a positive control are scores from vehicle-treated mice after LGE on d 0 (n = 11 eyes). *P < 0.001 compared with other groups. C) Cytotoxicity measured by Alamar Blue assay in FRT cells incubated with test compounds for 1 or 24 h (10% DMSO as positive control (n = 4). Means ± SEM. *P < 0.05 compared to untreated cells; P = 0.02 and 0.0006 for 1 and 24 h, respectively.

Figure 6.

Topical CFTR_act-K089 restores tear volume, preventing and reversing corneal epithelial disruption after lacrimal ablation. A) Left: basal tear volume after extraorbital LDC and LGE in BALB/c mice, comparing eyes treated with 0.1 nmol CFTR_act-K089 (n = 15 eyes) to vehicle (n = 11 eyes). Means ± sem. Tear volume was measured immediately before LDC/LGE, and then 1 h after the first daily dose on d 4, 10, and 14 after LGE. *P < 0.001. Right: corneal epithelial disruption after LDC/LGE measured by LG scoring on a 12-point scale in the same eyes. Means ± sem. *P < 0.001. B) Representative photographs of eyes before LDC/LGE (left) and on d 14 after LDC/LGE in vehicle-treated and CFTR_act-K089-treated eyes (right). C) Top: PAS staining in conjunctival fornices of vehicle-treated (left) and CFTR_act-K089-treated mice (right). Bottom: mean linear conjunctival goblet cell densities comparing mice treated with CFTR_act-K089 (n = 11 eyes) or vehicle (n = 10 eyes) for 14 d after LDC/LGE. Mean ± sem. **P < 0.01. D) Left: basal tear volume following LDC with CFTR_act-K089 (0.1 nmol; n = 6 eyes) or vehicle (n = 4 eyes) starting at d 5 after LDC. Means ± sem. *P < 0.001. Right: Corneal epithelial disruption after LDC in the same eyes. Means ± sem. *P < 0.001.