Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins

Abstract

Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC₅₀ down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC₅₀ down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.

Keywords: cholera; intestinal secretion; secretory diarrhea; traveler’s diarrhea.

Figure 1.

CFTR inhibition by (R)-BPO-27. A) Chemical structure of (R)-BPO-27. B) Short-circuit current in FRT cells expressing human wild-type CFTR showing responses to 100 μM CPT-cAMP (left) and 200 μM 8-Br-cGMP (right), with indicated concentrations of (R)-BPO-27 (representative of 3 experiments). C) Left: short-circuit in HBE cells in response to agonists and inhibitors that target key ion transport processes: 20 μM amiloride (ami), 20 μM forskolin (fsk), 10 μM CFTR_inh-172, and 100 µM ATP. Experiments were done in the absence and presence of (R)-BPO-27 (10 μM, 10 min pretreatment). Right: data summary (means ± se; n = 3). D) Left: short-circuit current in TMEM16A-expressing FRT cells in response to 100 µM ATP. Experiments were done in the absence and presence of (R)-BPO-27 (10 μM, 5 min pretreatment). Right: data summary (means ± se; n = 3). E) Short-circuit current in mouse midjejunal strips showing responses to 20 µM forskolin (left), 10 μg/ml STa toxin (right), and 1 μM (R)-BPO-27 (representative of 3 experiments).

Figure 2.

(R)-BPO-27 prevents fluid accumulation in cholera toxin-treated intestinal closed loops. A) Experimental protocol (left), loop weight/length ratio (middle), and representative photos (right) of loops treated with PBS or cholera toxin without or with (R)-BPO-27 (means ± se; n = 5–8 loops per group). B) (R)-BPO-27 dose-response study for experiments as in A (means ± se; n = 4–8 loops per group). C) Efficacy of orally administered (R)-BPO-27 (5 mg/kg) administered 60 min before surgery (means ± se; n = 3–7 loops per group). One-way ANOVA with post hoc Newman-Keuls multiple comparisons test. ***P < 0.001. ns, not significant.

Figure 3.

(R)-BPO-27 prevents fluid accumulation in STa toxin-treated intestinal closed loops. A) Experimental protocol (left), loop weight/length ratio (middle), and representative photos (right) of loops treated with PBS or STa toxin without or with (R)-BPO-27 (means ± se; n = 4 or 5 loops per group). B) (R)-BPO-27 dose-response study for experiments as in A (means ± se; n = 4 or 5 loops per group). One-way ANOVA with post hoc Newman-Keuls multiple comparisons test. **P < 0.01. ns, not significant.

Figure 4.

(S)-BPO-27 does not inhibit intestinal secretion, and (R)-BPO-27 does not inhibit intestinal absorption. A) Cholera toxin and STa toxin-induced intestinal secretion study done as in Figs. 2A and 3A but with (S)-BPO-27 (inactive enantiomer, 5 mg/kg, i.p.) (means ± se; n = 4–7 loops per group). One-way ANOVA with post hoc Newman-Keuls multiple comparisons test. B) Intestinal absorption study protocol (left), loop weight/length ratios at 0 and 30 min (middle), and percent intestinal absorption (right) in PBS-injected loops (means ± se; n = 4 loops per group); Student’s t test. ns, not significant.

Figure 5.

(R)-BPO-27 pharmacokinetics and toxicity. A) Standard serum concentration curve determined by LC-MS (left). Time course of serum (R)-BPO-27 concentration after bolus intraperitoneal or oral administration of 5 mg/kg (R)-BPO-27 at zero time (right) (means ± se; 3 mice per group). B) Cytotoxicity measured by Alamar Blue assay in FRT cells incubated with (R)-BPO-27 for 8 or 24 h (10% DMSO as positive control) (means ± se; n = 4 cultures). C) Body weight in mice receiving 5 mg/kg (R)-BPO-27 orally for 7 d (means ± se; n = 5 mice per group); Student’s t test. ns, not significant.

Figure 6.

(R)-BPO-27 inhibits CFTR-dependent swelling of enteroids generated from human jejunum and CFTR Cl⁻ current in planar enteroid cultures. A) Inhibition of forskolin-induced swelling of enteroids by (R)-BPO-27. Enteroids were incubated with (R)-BPO-27 for 10 min before addition of 5 µM forskolin. Enteroid swelling was quantified as areas of fluorescently stained enteroids as determined by confocal microscopy (means ± se; n = 8). B) Short-circuit current in planar cultures generated from the human enteroids. Cultures were treated with 10 µM forskolin, and then indicated concentrations of (R)-BPO-27 were added to both apical and basolateral solutions, followed by 25 µM CFTR_inh-172 added to the apical solution. Representative of 2 sets of studies.