Crofelemer, an antisecretory antidiarrheal proanthocyanidin oligomer extracted from Croton lechleri, targets two distinct intestinal chloride channels

Abstract

Crofelemer, a purified proanthocyanidin oligomer extracted from the bark latex of Croton lechleri, is in clinical trials for secretory diarrheas of various etiologies. We investigated the antisecretory mechanism of crofelemer by determining its effect on the major apical membrane transport and signaling processes involved in intestinal fluid transport. Using cell lines and measurement procedures to isolate the effects on individual membrane transport proteins, crofelemer at 50 microM had little or no effect on the activity of epithelial Na(+) or K(+) channels or on cAMP or calcium signaling. Crofelemer inhibited the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel with maximum inhibition of approximately 60% and an IC(50) approximately 7 microM. Crofelemer action at an extracellular site on CFTR produced voltage-independent block with stabilization of the channel closed state. Crofelemer did not affect the potency of glycine hydrazide or thiazolidinone CFTR inhibitors. Crofelemer action resisted washout, with <50% reversal of CFTR inhibition after 4 h. Crofelemer was also found to strongly inhibit the intestinal calcium-activated Cl(-) channel TMEM16A by a voltage-independent inhibition mechanism with maximum inhibition >90% and IC(50) approximately 6.5 microM. The dual inhibitory action of crofelemer on two structurally unrelated prosecretory intestinal Cl(-) channels may account for its intestinal antisecretory activity.

Figures

Fig. 1.

Cellular mechanisms of intestinal fluid secretion by enterocytes, showing chloride secretion through apical membrane chloride channels. See the Introduction for a further explanation.

Fig. 2.

Crofelemer reduces Cl− secretion in T84 human intestinal cells in response to cAMP and calcium-elevating agonists. A, chemical structure of crofelemer (see Materials and Methods for explanation) (Ubillas et al., 1994). B, short-circuit current in T84 cells after activation of Cl− secretion by forskolin (10 μM), ATP (100 μM), or thapsigargin (1 μM). Indicated concentrations of crofelemer were added to the luminal bathing solution. Where indicated, cells were pretreated with 20 μM CFTRinh-172 to inhibit CFTR Cl− current. C, short-circuit current measurements showing CFTR (left)- and CaCC (right)-dependent Cl− current in the presence of crofelemer (50 μM) added to the basolateral bathing solution 10 min before measurements. CFTR was inhibited by pretreatment with CFTRinh-172 (20 μM) for measurement of ATP-induced CaCC activation.

Fig. 3.

Crofelemer inhibition of CFTR Cl− conductance. A, apical membrane current in CFTR-expressing FRT cells after permeabilization with amphotericin B and in the presence of a transepithelial Cl− gradient (apical [Cl−], 75 mM; basolateral [Cl−], 150 mM). CFTR Cl− conductance was activated by 100 μM CPT-cAMP followed by the addition of indicated concentrations of crofelemer to the luminal solution. B, crofelemer concentration inhibition of CFTR Cl− current measured at 20 min after crofelemer application (S.E., n = 3–5). Data shown for experiments as in A (○) and with reversed Cl− gradient (apical [Cl−], 150 mM; basolateral [Cl−], 75 mM) (●).

Fig. 4.

Characterization of crofelemer inhibition of CFTR Cl− conductance. A, crofelemer inhibition of CFTR after different agonists including forskolin (20 μM) and apigenin (100 μM). B, slow reversibility of crofelemer inhibition of CFTR. Where indicated, crofelemer was added, the apical solution was washed extensively, and CPT-cAMP was readded. C, investigation of possible synergy/competition of crofelemer with small-molecule CFTR inhibitors. Left, apical membrane current after CFTR activation by CPT-cAMP and inhibition by CFTRinh-172 or GlyH-101. Right, crofelemer (50 μM) was added to inhibit CFTR Cl− current by ∼50 to 60%, followed by indicated concentrations of CFTRinh-172 or GlyH-101.

Fig. 5.

Patch-clamp analysis of crofelemer inhibition of CFTR. A, left, whole-cell CFTR current recorded at a holding potential at 0 mV and pulsing to voltages between ±100 mV in steps of 20 mV in the absence and presence of 50 μM crofelemer. CFTR was stimulated by forskolin. Right, I-V plot of mean currents at the middle of each voltage pulse from experiments as in A (S.E., n = 3). Fitted IC50 = 6.5 μM. B, left, single-channel recordings were done in the cell-attached configuration. CFTR was activated by 10 μM forskolin and 100 μM IBMX. Pipette potential was +80 mV. Right, summary of crofelemer effect on CFTR channel Po, mean open time, and mean closed time (S.E., n = 3–4; ∗, P < 0.05). o, open channel state; c, closed channel state.

Fig. 6.

Crofelemer inhibition of calcium-activated Cl− channels. A, apical membrane current in TMEM16A-expressing FRT cells in the presence of a transepithelial Cl− gradient (apical [Cl−], 70 mM; basolateral [Cl−], 140 mM). B, crofelemer concentration-dependence of TMEM16A Cl− current inhibition. C, whole-cell TMEM16A current recorded at a holding potential of 0 mV and pulsing to voltages between ± 100 mV in steps of 20 mV in the absence and presence of 10 μM crofelemer. TMEM16A was stimulated by 100 μM ATP. D, I-V plot of mean currents (at the middle of each voltage pulse). Mean currents were normalized as current densities (measured in picoamperes per picofarads).

Fig. 7.

Little or no effect of crofelemer on apical membrane cation channels and intracellular cAMP and calcium signaling. A, left, short-circuit current in primary cultures of CFTR-deficient human bronchial epithelial cells without versus with pretreatment with 50 μM crofelemer in the luminal solution. Where indicated, amiloride (10 μM) and UTP (100 μM) were added. Right, summary of differences in short-circuit current after amiloride and UTP additions (S.E., n = 3; ∗, P < 0.05). B, apical membrane K+ current in human bronchial epithelial cells after basolateral membrane permeabilization with 20 μM amphotericin B and in the presence of a K+ gradient (apical [K+], 5 mM; basolateral [K+], 150 mM). C, cAMP levels in T84 cell homogenates under basal conditions and at 10 min after treatment with 20 μM forskolin. Differences ± crofelemer were not significant. D, calcium signaling measured by fura-2 fluorescence in T84 cells under basal conditions and after ATP addition (100 μM). Where indicated, cells were pretreated with 50 μM crofelemer. Inset, peak ATP increase in fura-2 fluorescence ratio (S.E., n = 4). Difference was not significant.