Abstract:Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecu… Show more
“…In air-exposed mice, roflumilast more than doubled the water content of stool specimen (roflumilast, 3.1 ± 0.2 vs. vehicle, 1.4 ± 0.1, P < 0.0001), corroborating our ex vivo findings [ 20 ]. Interestingly, exposure to cigarette smoke increased the wet/dry ratio of specimen to a level (2.6 ± 0.2) beyond which roflumilast had minimal additional effect (2.8 ± 0.3); given that the use of oral CFTR inhibitors that are not systemically absorbed has been proposed as an approach for offsetting roflumilast-associated diarrhea [ 29 , 30 ], this finding presents the possibility that this strategy may be an effective approach worth evaluating prospectively, even in the background of cigarette smoking. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Noting the evidence associating cigarette smoke with impaired CFTR function, even in the intestine [ 9 ], it is likely that this effect was mediated through a CFTR-independent mechanism. Nevertheless approaches currently being developed to treat diarrhea via CFTR inhibition by oral, non-bioavailable molecules [ 29 , 30 ] may be of potential benefit in a subpopulation of patents who are cigarette smokers and experience diarrhea, particularly those who are intolerant of roflumilast, although further studies are needed to confirm these relationships.…”
BackgroundDysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction.MethodsA/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea.ResultsAcute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, −2.0 ± 0.4 mV, 131.3 ± 29.3 μA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 μA/cm2, P < 0.01 vs. vehicle −0.3 ± 0.7 mV, 10.4 ± 7.0 μA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, −15.22 ± 2.7 mV vs. air, −14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect.ConclusionsRoflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.
“…In air-exposed mice, roflumilast more than doubled the water content of stool specimen (roflumilast, 3.1 ± 0.2 vs. vehicle, 1.4 ± 0.1, P < 0.0001), corroborating our ex vivo findings [ 20 ]. Interestingly, exposure to cigarette smoke increased the wet/dry ratio of specimen to a level (2.6 ± 0.2) beyond which roflumilast had minimal additional effect (2.8 ± 0.3); given that the use of oral CFTR inhibitors that are not systemically absorbed has been proposed as an approach for offsetting roflumilast-associated diarrhea [ 29 , 30 ], this finding presents the possibility that this strategy may be an effective approach worth evaluating prospectively, even in the background of cigarette smoking. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Noting the evidence associating cigarette smoke with impaired CFTR function, even in the intestine [ 9 ], it is likely that this effect was mediated through a CFTR-independent mechanism. Nevertheless approaches currently being developed to treat diarrhea via CFTR inhibition by oral, non-bioavailable molecules [ 29 , 30 ] may be of potential benefit in a subpopulation of patents who are cigarette smokers and experience diarrhea, particularly those who are intolerant of roflumilast, although further studies are needed to confirm these relationships.…”
BackgroundDysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction.MethodsA/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea.ResultsAcute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, −2.0 ± 0.4 mV, 131.3 ± 29.3 μA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 μA/cm2, P < 0.01 vs. vehicle −0.3 ± 0.7 mV, 10.4 ± 7.0 μA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, −15.22 ± 2.7 mV vs. air, −14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect.ConclusionsRoflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.
“…STa is an enterotoxin that causes gastrointestinal electrolyte imbalance characterized by a higher Cl - release to the gastrointestinal lumen, a phenomenon that ends in diarrhoea in humans [ 1 , 3 – 5 ]. One of the potential mechanisms for these adverse effects of STa is a mucosal alkalization due to lower activity of plasma membrane mechanisms involved in maintaining transmembrane distribution of H + , including NHEs activity [ 10 , 11 ].…”
Section: Discussionmentioning
confidence: 99%
“…ETEC colonizes host intestines and releases heat-labile and/or heat-stable (STa) enterotoxins. STa causes secretory diarrhoea and is responsible for about half of all ETEC–related diarrhoeal diseases, including traveller’s diarrhoea and epidemic diarrhoea of the newborn [ 1 – 5 ].…”
The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (J
H
+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J
H
+ (~63%), without altering basal pHi (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and J
H
+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J
H
+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.
“…Pharmacological activation of CF-causing mutant CFTRs has emerged as a major therapeutic strategy in CF (2), and activation of wild-type CFTR in subjects without CF has potential utility for the treatment of constipation (3), dry eye (4), and potentially inflammatory lung diseases (5). Pharmacological inhibition of CFTR has predicted utility in the treatment of enterotoxin-mediated secretory diarrheas, including cholera and traveler's diarrhea, and of autosomal dominant polycystic kidney disease (ADPKD) because CFTR activation causes intestinal fluid secretion in certain diarrheas (6)(7)(8) and fluid accumulation in renal cysts in ADPKD (9)(10)(11)(12).…”
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 IC down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC 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.
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