Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy

<div><p>Background and Aims</p><p>Morbidity and mortality from acute diarrheal disease remains high, particularly in developing countries and in cases of natural or man-made disasters. Previous work has shown that the small molecule clotrimazole inhibits intestinal Cl^- secretion by blocking both cyclic nucleotide- and Ca²⁺-gated K⁺ channels, implicating its use in the treatment of diarrhea of diverse etiologies. Clotrimazole, however, might also inhibit transporters that mediate the inwardly directed electrochemical potential for Na⁺-dependent solute absorption, which would undermine its clinical application. Here we test this possibility by examining the effects of clotrimazole on Na⁺-coupled glucose uptake.</p><p>Materials and Methods</p><p>Short-circuit currents (Isc) following administration of glucose and secretagogues were studied in clotrimazole-treated jejunal sections of mouse intestine mounted in Ussing chambers.</p><p>Results</p><p>Treatment of small intestinal tissue with clotrimazole inhibited the Cl^- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP) or Ca²⁺-agonist carbachol in a dose-dependent fashion. A dose of 30 μM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37). There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90).</p><p>Conclusion</p><p>Treatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca²⁺-dependent agonists as expected, but did not affect Na⁺-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas.</p></div

CLT blocks the action of secretagogues without impairing glucose uptake.

<p><b>(A)</b> Isc registration of vehicle (n = 6) or CLT-treated (n = 5) tissues that were sequentially challenged with 10mM glucose apically (t = 10), and 7.5 nM VIP (t = 25) and 20 μM carbachol (t = 40) basolaterally. (<b>B-D</b>) Summary statistics of the maximal recorded change in Isc (ΔIsc) within 10 minutes of challenge with the investigated compound from baseline Isc before challenge. Pooled data from 2 independent experiments. Error bars represent the SEM. *p<0.05; ***p<0.001 as assessed by student t-test.</p

Clotrimazole inhibits the effect of secretagogues in a dose-dependent fashion.

<p>Jejunal tissue preparations were apically and basolaterally treated with 5, 30, and 150 μM clotrimazole (CLT) or vehicle control for 20 minutes in Ussing chambers and subsequently challenged with the cAMP-dependent secretagogue VIP (7.5 nm) or the Ca²⁺-dependent secretagogue carbachol (20 μM). Short-circuit current (Isc) registration and statistical analysis following VIP (<b>A</b>) and carbachol (<b>B</b>) administration to basolateral chamber at t = 5 and t = 1 respectively show dose-dependent inhibition of secretory Isc by CLT. Pooled data from 3 independent experiments, with a total of 4 intestinal sections per condition. Error bars represent the SEM. *p<0.05; ** p<0.01; ***p<0.001; NS: not significant as determined by ANOVA with Bonferroni’s multiple comparison test.</p

Peripheral blood dendritic cells, monocytes, basophils, and neutrophils express FcεRI, CD23 and receptor-bound IgE at the cell surface.

<p>Number of positive cells, median and range of % positive cells among the respective population is shown. If n = 1, the % positive cells in this patient is displayed.</p

Clotrimazole treatment does not inhibit the function of SGLT1.

<p><b>(A)</b> Equivalent changes in Isc following apical administration of 30 mM 3-O-methyl glucose (3-OMG) rule out an inhibitory effect on SGLT1 from 30 μM CLT. (<b>B</b>) Phloridzin (PHL)-mediated SGLT1 blockade decreases Isc changes in response to 3-O-methyl glucose but does not affect Cl^- secretion in response to VIP, with individual data points at t = 5 depicted in (<b>C</b>). (<b>D</b>) ΔIsc elicited by apical administration of 10 mM glucose is partially blocked by PHL treatment. Pooled data from 2 independent experiments in A and single experiments in B-D. Error bars represent SEM. *p<0.05; **p<0.01; NS not significant as assessed by student t-test on the ΔIsc following addition of described stimulus at the indicated time points.</p

Peripheral blood cells carry IgE, even in the absence of elevated serum IgE.

<p>The percentages of IgE-loaded FcεRI+ peripheral blood cells did not significantly differ between patients with elevated versus normal serum IgE (Mann-Whitney-U-test). However, some patients with normal serum IgE showed high levels of cell-bound IgE.</p

Serum IgE enhances FcεRI on dendritic cells and monocytes from patients with elevated IgE.

<p><b>A</b>. Cell surface levels of FcεRI on dendritic cells and monocytes correlate with serum IgE in children with elevated IgE. <b>B</b>. No correlation was found in patients with normal serum IgE. Data points represent percent (%) positive cells among cell type after subtraction of isotype control background in individual patients.</p

Cell surface-bound IgE correlates with serum IgE and IgE-receptor expression.

<p><b>A</b>. Surface-bound IgE in FcεRI+ DC, monocytes, and neutrophils correlates with serum IgE. <b>B</b>. FcεRI-expression correlates with surface-bound IgE in FcεR+ DC, monocytes, and basophils, CD23-expression correlates with surface-bound IgE in CD23+ DC and monocytes (Spearman rank correlation, respectively).</p><p>*p<0.05.</p

FcεRI expression increases with age in dendritic cells and monocytes.

<p>Data points represent percent (%) positive cells among cell type after subtraction of isotype control background in individual children.</p

Unexpected apparent molecular mass of ISG15VS-DUB adducts is caused by unusual behavior in SDS-PAGE.

<p>(A) Plot showing the ratio of observed versus expected ISG15VS-DUB adduct size of USP2, USP5, USP13, USP14 and USP18 in SDS-PAGE (8%) over the size of the unmodified DUBs. (B) Mutation of the catalytic cysteine residue to serine (C114S) in USP14 abolishes its reactivity toward UbVME and ISG15VS. When stored for longer periods at room temperature, the probes polymerize covalently, presumably by formation of secondary amine bonds between internal lysine residues and the reactive Michael acceptor at the C-terminus, thus resembling isopeptide-linked polyubiquitin. Such polymeric probes of UbVME likely caused the additional high-molecular mass adducts observed for USP14. Note that the smallest version of these adducts (a UbVME dimer) has a maximum electrophoretic mobility similar to that of the diubiquitin-like ISG15VS when complexed to USP14. The absence of any adducts in the C114S mutant of USP14 excludes the possibility of multiple binding sites for the probes. (C) Purified recombinant USP14 labels with UbVME and ISG15VS and results in the same abnormal mobility shift for ISG15VS-USP14 as seen in the IVT labeling experiments. (D) MALDI-TOF mass spectroscopic analysis of USP14 after incubation with ISG15VS. As described above for UbVME, ISG15VS also engages in internal polymerization. Molecular masses consistent with tri- and tetrameric ISG15VS are marked in this spectrogram by the numbers in superscript. Monovalently modified USP14 results in an adduct of predicted size, indicated with a red arrow. This complex is unique to the mixture containing both USP14 and ISG15VS, and is absent in the mass spectra of either component alone (data not shown).</p