1 Department of Pediatric Gastroenterology and the 2 Cystic Fibrosis/Pulmonary Research and Clinical Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599; and 3 SHAMAN Pharmaceuticals Inc., South San Francisco, California 94080
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ABSTRACT |
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We have identified an agent (SP-303) that shows
efficacy against in vivo cholera toxin-induced fluid secretion and in
vitro cAMP-mediated Cl
secretion. Administration of cholera toxin to adult mice results in an
increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals,
respectively). This elevation in FA induced by cholera toxin was
significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment.
Moreover, when SP-303 was administered 3 h after cholera toxin, a
dose-dependent inhibition of FA levels was observed with a half-maximal
inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or
T84 monolayer preparations, SP-303 had a significant
effect on both basal current and forskolin-stimulated Cl
current. SP-303 also
induced an increase in resistance that paralleled the observed decrease
in current. These data suggest that SP-303 has an inhibitory effect on
cAMP-mediated Cl
and fluid
secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.
cystic fibrosis transmembrane conductance regulator; secretory diarrhea; chloride channel
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INTRODUCTION |
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SECRETORY DIARRHEA REMAINS one of the major causes of
infant morbidity and mortality around the world, accounting for over a
billion episodes per year in children under 5 years of age (13). In
comparison to other mechanisms of diarrheal disease, secretory diarrheas caused by bacterial enterotoxins [e.g.,
Vibrio cholerae toxin and
Escherichia coli, heat stable (ST),
heat labile (LTa), and LTb toxins] are not
associated with severe intestinal histopathology. Rather, secretory
diarrheas appear to function by stimulating transepithelial
Cl secretion, thereby
increasing the osmotic impetus for fluid secretion (4). Cholera toxin
(CT) is likely the most recognizable enterotoxin that causes secretory
diarrhea. After colonization of the small intestine by
Vibrio cholerae, binding of the
cholera enterotoxin to the intestinal enterocyte leads to
ADP-ribosylation of the
-subunit of a stimulatory G protein,
Gs
(7, 11). This covalent modification activates adenylate cyclase for the lifetime of the enterocyte during its migration up the villus axis. As a consequence of
this irreversible elevation of cAMP, an apical membrane
Cl
channel is activated
that results in a voluminous
Cl
and fluid secretion that
can be fatal if untreated (4, 7).
Cystic fibrosis (CF) is a disease characterized by defective
cAMP-regulated Cl secretion
of the apical membrane of epithelial cells (12, 14). Cloning of the CF
gene (10, 16) and expression studies of the protein product, the cystic
fibrosis transmembrane conductance regulator (CFTR), reveal that it is
a cAMP-regulatable Cl
channel (1, 2, 18). CFTR is widely expressed in many epithelial tissues
and is prevalent in the mammalian small intestinal epithelium in both
crypt and villus cells (20). Gene-targeting approaches have
successfully been used to create several CF mouse models (reviewed in
Ref. 9), all of which show that a primary pathophysiology occurs in the
gastrointestinal tract. This pathophysiology can be explained by the
finding that CFTR is the predominant mechanism for apical
Cl
secretion in the murine
gastrointestinal tract (5). Thus a functional absence of CFTR in the
CFTR(
/
) mouse results in a severely dehydrated intestinal
epithelium that is the catalyst for constrictions, obstructions, and
ultimately death (17).
SP-303 is derived from the latex of the plant Croton
lechleri and comprises a mixture of naturally occurring
proanthocyanidin oligomers with an average molecular mass of 2,200 Da
(21). This naturally occurring latex has been used by the indigenous
people of South America to treat various kinds of watery diarrheas,
including diarrhea caused by cholera. In this study, we examine the
ability of SP-303 to inhibit CT-induced fluid secretion in the murine intestine. We also test whether this compound is capable of inhibiting Cl secretion across a human
intestinal epithelial cell monolayer.
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MATERIALS AND METHODS |
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Fluid accumulation. Fluid accumulation (FA) was measured using the sealed adult mouse model for secretory diarrhea as described previously (8, 15). Briefly, 8- to 10-wk-old C57Bl/6 mice of either sex with body masses that ranged from 12.1 to 36.0 g were used. Initially, mice were given single doses of SP-303 dissolved in 7% wt/vol NaHCO3 delivered by an orogastric feeding tube, followed immediately by 10 µg CT (this is referred to as simultaneous addition). Subsequent experiments involved administration of 10 µg CT at time 0 followed by administration of varying doses of SP-303 at 3 h (one set of experiments, involving SP-303 prepared as an enteric-coated bead). Under either of the experimental conditions, mice were killed 6 h after the administration of CT. The entire small intestine, from the pylorus to the cecum, was isolated with care to avoid tissue rupture and loss of fluid. The attached mesentery and connective tissues were removed, and the mass of the tissue and enclosed fluid was determined. The tissue was then opened longitudinally, the fluid was removed, and the tissue was patted dry. The mass of the dry tissue was then reweighed. FA was measured as a ratio of the mass of accumulated fluid in the small intestine and cecum vs. the mass of the small intestine minus the mass of the fluid. Both the CT dose administered and the time of incubation were selected to ensure that a maximal amount of fluid would be secreted into the intestinal lumen. Data were analyzed to determine whether SP-303 administration resulted in a statistically significant reduction in CT-induced FA ratios compared with CT-treated animals given only NaHCO3 as a control solution. Baseline (e.g., nonsecretory) animals were administered a bolus of 7% NaHCO3 solution in the absence of CT, and FA values were measured 6 h later.
Cell culture.
Human colonic epithelial cell lines, either Caco-2 or T84, were grown
in either DMEM (Caco-2) or DMEM-Ham's F-12 (T84) supplemented with 8 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 10-15% FCS, in
an atmosphere of 95% air and 5%
CO2. Cell monolayers were plated
at high density (2 × 104
cells per mm2) onto tissue
culture inserts (collagen matrix supports with a 4.5-mm plating
diameter) as described previously (6). For all experiments, cells were
evaluated for confluence by daily monitoring of transepithelial
resistance (Rt)
and potential difference (PD). Only monolayers generating at least a
0.5 mV PD and a 150 · cm2
resistance were used for Ussing chamber studies.
Ussing chamber studies.
Electrical measurements, PD, and short-circuit current
(Isc) were made
on cultured epithelial cell monolayers mounted in Ussing chambers (4).
Unless otherwise stated, the bathing medium used for all studies was a
standard Krebs-Ringer bicarbonate solution (KRB) on the basolateral
side and a modified KRB, high-K+
and low-Cl solution
(gluconate was substituted for
Cl
) on the apical side
(6). In some experiments, an apical
Na+-free solution
(N-methyl-D-glucamine
was substituted for Na+) was
used to eliminate any contribution of absorptive
Na+ channels. In these
experiments, ion solutions were balanced with the exception of
Na+ (140 mM
Na+ in the basolateral solution
and 0 mM Na+ in the apical
solution). All bathing solutions were bubbled with 95%
O2-5%
CO2 and maintained at 37°C. PD
was clamped to zero, and the
Isc was
continuously displayed on a pen recorder.
Rt was calculated
from the magnitude of the current deflections in response to a voltage
pulse imposed on short-circuited cell sheets every 60 s with a duration
of 0.5 s. Equivalently, electrometer output was digitized online, and
Isc,
Rt, and
calculated transepithelial potential were displayed on a video monitor
and stored on a computer hard drive. Drugs were added from concentrated
stock solutions to either apical and/or basolateral bathing
solutions. All compounds were added cumulatively, i.e., drugs were not
removed before addition of subsequent doses or compounds. Data are
expressed as means ± SE for n (no.
of experiments).
cAMP measurements. T84 cell monolayers were plated and cultured on tissue culture inserts as described for Ussing chamber studies. Cells were bathed in KRB apical and basolateral solutions, and CT or SP-303 was administered from concentrated solutions to the apical bath. T84 cells were administered a single dose of 10 µg CT at time 0 followed by 300 µM SP-303 3 h later. Bathing solutions were removed, and 6% ice-cold TCA was added to the cells at hour 6 to stop the reaction. Precipitated protein and supernatant were collected and centrifuged (1,000 g for 5 min). Ether extracts of the supernatants were reconstituted and assayed for cAMP content by an acetylated RIA kit (Biomedical Technologies, Stoughton, MA). All samples were measured in duplicate and titrated to lie within the linear range of the standard curve. Student's t-test was used to determine statistically different means.
Materials. All biochemicals used were obtained from commercial sources and were of tissue culture grade or better. SP-303 was prepared as described previously and verified to be at least 95% pure by HPLC (21).
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RESULTS |
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We have previously shown that CT-induced fluid and
Cl secretion is directly
proportional to the expression level of the CFTR Cl
channel (8). In this
study, we investigate the effects of SP-303 on CT-induced FA in the
small intestine of mice expressing normal levels of CFTR. Control mice
treated with only a bolus of 7%
NaHCO3 solution in the absence of
either CT or SP-303 show an FA ratio of 0.63 ± 0.07 (Fig.
1, dashed line in
A and
C). CT (10 µg)-treated animals
show an FA level (1.86 ± 0.2) that is nearly three times greater
than control values. Simultaneous administration of SP-303 with CT
results in an inhibition of FA in a concentration-dependent manner
(Fig. 1A). Gavage administration
of 100 mg/kg SP-303, simultaneously with CT treatment, restores FA to
near normal levels (0.70 ± 0.19). To exclude the possibility that
SP-303 may be either interacting with CT or preventing binding of CT to
the ganglioside receptor, we studied SP-303-mediated inhibition of FA
in mice prestimulated by CT. Mice that received a dose of SP-303 3 h
after exposure to CT showed a significant decrease in FA ratios (Fig.
1B). The dose-response curve showed
inhibition with an apparent half-maximal inhibitory concentration
(IC50) of ~10 mg/kg. The
highest SP-303 doses administered brought about a nearly complete
inhibition of CT-induced FA levels [i.e., 50 mg/kg SP-303-treated
mice had FA values that were not significantly different from untreated control FA values (0.78 ± 0.08 vs. 0.63 ± 0.07)]. In an
attempt to maximize efficient delivery of SP-303 to the small
intestinal lumen and in anticipation of future clinical trials, SP-303
was encapsulated in an alkali-soluble, enteric-coated bead and FA ratios were measured (Fig. 1C).
Administration of SP-303 encapsulated in this formulation was likewise
capable of significantly inhibiting CT-induced FA (0.75 ± 0.10 vs.
1.51 ± 0.08 for CT and 100 mg/kg SP-303 vs. treatment with CT
alone, respectively).
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Secretory diarrhea as induced by CT is the result of irreversible
activation of adenylate cyclase, leading to elevation of cAMP and
finally stimulation of Cl
secretion. Thus we sought to determine if the observed SP-303 inhibition of FA was due to a metabolic inhibition of cellular cAMP
levels. We measured the effect of SP-303 on cellular cAMP levels in
confluent T84 cell monolayers. Administration of CT (10 µg/ml)
resulted in a large increase in cAMP levels sampled 6 h after treatment
(1,031.4 ± 64 vs. 4.97 ± 0.3 pmol/ml for CT vs. control,
respectively; n = 6 for each
condition). Addition of 300 µM SP-303 neither significantly reduced
the CT-elevated cAMP levels (1,031.4 ± 64 vs. 972.5 ± 77 pmol/ml for CT vs. CT and SP-303, respectively;
P = 0.57) nor reduced the
non-CT-treated basal cAMP levels (4.97 ± 0.3 vs. 5.51 ± 0.3 pmol/ml for control vs. control and SP-303, respectively;
P = 0.24).
The lack of an effect of SP-303 on cAMP levels suggests that the
compound is acting at a site distal to elevation of cAMP (i.e., the
CFTR Cl channel itself or a
regulator of the channel). We therefore investigated the effects of
SP-303 on the basal
Isc of Caco-2
epithelial cell monolayers as a measure of
Cl
secretion (Fig.
2). Sequential addition of SP-303 to the
solution bathing the apical membrane inhibited the basal
Isc by nearly 50%. Moreover, subsequent addition of forskolin (an activator of
adenylate cyclase) resulted in a relatively small
Cl
secretory response (Fig.
2A). Forskolin-stimulated currents
had a mean of 16.9 ± 2.5 µA/cm2
(n = 8) after exposure to
300 µM SP-303, whereas forskolin-stimulated currents in monolayers
not exposed to SP-303 showed a mean of 38.3 ± 4.3 µA/cm2
(n = 16). Inhibition of
basal Isc was
seen at all doses of SP-303, with maximal effects observed following
administration of 300 µM SP-303 (Fig.
2B). Importantly, increasing doses
of SP-303 resulted in an increase in monolayer resistance that
correlated with the decrease in
Isc (300 µM
SP-303 showed an increase in resistance of 39 ± 7% of
basal values, n = 9).
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To further define the effect of SP-303 on cAMP-mediated
Cl secretion, we examined
the ability of SP-303 to inhibit the maximum Isc stimulated by
forskolin in Caco-2 cells (Fig. 3).
Cl
secretion maximally
stimulated by the addition of forskolin was inhibited in a stepwise
manner by sequential additions of SP-303 to the apical solution (Fig.
3A). The forskolin-stimulated
current, if untreated, was previously shown (6) to be relatively stable for up to 45 min after stimulation. Maximal inhibition of the forskolin-stimulated
Isc was achieved
by the addition of 300 µM SP-303 (Fig.
3B). The IC50 for SP-303
was ~50 µM. Bumetanide, an inhibitor of the basolateral
Na+-K+-2Cl
cotransporter responsible for
Cl
entry, decreased the
residual Isc to
prestimulated levels.
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To determine that the effect of SP-303 was not restricted to a single
intestinal epithelial cell line, we also studied inhibition of
Cl secretion in another
colonic cell line, T84. The T84 cell line is known to express CFTR at
relatively high levels, and, when studied as a polarized monolayer in
Ussing chambers, cAMP causes a significant increase in
Isc that is
primarily due to Cl
secretion (19). Moreover, by bathing the apical surface of the T84
monolayers in an Na+-free
solution, we more effectively translate
Isc into a direct measurement of apical Cl
secretion. Under these conditions, addition of 500 µM SP-303 to the
apical solution inhibited nearly all the
Isc stimulated by
forskolin (mean inhibition of 88.7%;
n = 8) (Fig.
4).
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SP-303 did not have any effect on basal or forskolin-stimulated
Isc measured in
Caco-2 monolayers exposed to bilateral
Cl-free solutions (data not
shown), although under these conditions basal currents were decreased
before SP-303 addition. Basolateral addition of SP-303 to T84
monolayers was used as an assessment of nonspecific (metabolic)
inhibitory effects of SP-303. T84 monolayers were bathed in bilateral
KRB and exposed to 50 µM SP-303 in the basolateral bath followed by
apical addition of forskolin (5 µM). Basolateral addition of SP-303
neither had an effect on basal current (28.4 ± 2.4 vs. 27.4 ± 1.1 µA/cm2 before and after
exposure to SP-303, respectively; n = 3) nor did it significantly blunt the subsequent increase mediated by forskolin (
Isc
of 103.7 ± 11.4 vs. 119 ± 13.8 µA/cm2 with or without
pretreatment by SP-303, respectively;
n = 3). A further addition of 50 µM
SP-303 to the basolateral bath (total concentration = 100 µM) did not
have an effect on the forskolin-stimulated current. We were not able to
reverse the inhibitory effect of apical SP-303 on forskolin-stimulated
current in T84 monolayers. After stimulation of
Isc by forskolin,
addition of 100 µM SP-303 to the apical solution resulted in the
expected inhibition of current as previously reported in Fig.
3A. The apical bath solution was then
replaced with identical Ringer solution devoid of SP-303. This maneuver
did not restore
Isc levels (68.2 ± 7.8 vs. 67.8 ± 10.1 µA/cm2,
Isc measured
after inhibition by SP-303 and after bath replacement of SP-303,
respectively; n = 8).
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DISCUSSION |
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We have described effects of SP-303 on CT-induced fluid secretion in an
in vivo mouse model and also on cAMP-mediated
Cl secretion measured
across epithelial cell monolayers. The animal studies demonstrate a
potent, nearly complete inhibition of fluid secretion when SP-303 is
applied either early (simultaneous to toxin delivery) or late (3 h
after CT delivery, when cAMP levels are maximal and
Cl
secretion is strongly
stimulated; Ref. 8). Interestingly, SP-303 was effective regardless of
when it was delivered, suggesting that this agent may have utility as
either a postsymptomatic antidiarrheal or as a prophylactic. It remains
to be seen if SP-303 truly can function as a prophylactic agent and
also how long before the introduction of CT is SP-303 capable of
maintaining a protective effect.
Because SP-303 was shown to be efficacious 3 h after administration of
CT, it follows reasonably that SP-303 does not interfere with CT
binding, internalization, or cAMP elevation. We did, however, verify
that SP-303 had no effect on either basal or elevated cAMP levels
measured in T84 cells and thereby focused on a more direct effect on
Cl secretion. Furthermore,
preliminary studies in humans have not been able to detect SP-303 in
plasma following oral administration, suggesting little or no systemic
absorption (SHAMAN Pharmaceuticals, unpublished observations).
Consistent with this hypothesis of little or no transcellular flux of
SP-303 is the finding that administration of SP-303 to the basolateral
solution had no significant effect on either basal or
forskolin-stimulated currents. Importantly, we did not observe an
effect of SP-303 on other non-CFTR transport processes (e.g.,
bumetanide-sensitive
Na+-K+-2Cl
cotransport or carbachol-mediated
K+ secretion; unpublished
observations). These data suggest that administration of SP-303 by
gavage is likely to have its effects at the apical membrane.
We have observed that the magnitude of
Cl secretion mediated by
forskolin is at least partially dependent on the magnitude of the basal
current. Because CFTR is the primary mechanism for Cl
secretion in both Caco-2
and T84 cells, it follows reasonably that channels that are already
open and maximally active in the basal condition are not likely to be
further activated by forskolin. Thus it is not surprising that we have
observed effects of SP-303 on both basal and forskolin-stimulated
Cl
currents.
These data suggest that SP-303 may be acting at the site of
Cl secretion, perhaps by
directly inhibiting CFTR or by altering regulation of this channel.
Interestingly, the active compound contains several exposed hydroxyl
and carboxylic acid residues that might be able to penetrate the mouth
of the Cl
channel and
occlude the pore and would thereby effectively inhibit various
secretory diarrheas. It is still possible, however, that SP-303 may be
exerting a general metabolic effect that may deplete cytosolic ATP
levels and prevent normal gating of CFTR or other channels. Direct
effects of SP-303 on the CFTR
Cl
channel will require
more mechanistic studies, involving patch-clamp protocols to
definitively determine if there is a direct effect.
Although these data suggest an effect on cAMP-mediated
Cl conductance, it is not
yet clear if this is a specific effect. Previous studies have
demonstrated that CFTR is the predominant
Cl
channel in the apical
membrane of the murine small intestine (5), but, in other epithelial
tissues, SP-303 may interact with other
Cl
channels, i.e., volume,
voltage, or intracellular
Ca2+-activated
Cl
channels. If SP-303 does
show high specificity to CFTR, by either inhibition and/or
binding, this compound will be of great utility as a tool in
understanding CFTR structure-function relationships, aside from its use
as an antidiarrheal drug.
Although cholera is the classic example of a secretory diarrhea, other bacterial toxins such as E.coli ST and LT are also capable of causing fluid secretion. As shown previously, agents such as E.coli STa, regardless of initial second messenger activation, ultimately result in elevation of cAMP and stimulation of CFTR (3). Therefore, a specific inhibitor acting directly at CFTR could prove to be very useful as it would inhibit the final step associated with most if not all known bacterial agents causing secretory diarrhea.
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ACKNOWLEDGEMENTS |
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We thank Drs. Barb Grubb, Jack Stutts, Ric Boucher, James Pennington, and Steven Porter for helpful discussions and suggestions. We also thank Pauline Blonigen, Dr. Rob Sweezey, J. Hung, Dr. M. F. King, and Dr. A. Sabouni for technical assistance and Neha Mehta and the Center for Gastrointestinal Biology and Disease (Univ. of North Carolina) for cAMP analyses (National Institutes of Health Grant P30-DK-34987). This work would not have been possible without the efforts of the ethnobotanical team, led by Dr. Steven King, in collaboration with the indigenous people of Latin America whose knowledge of the healing properties of Croton lechleri helped in the identification of this compound.
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FOOTNOTES |
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This work was supported by a contract awarded to S. E. Gabriel by SHAMAN Pharmaceuticals Inc.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: S. E. Gabriel, Dept. of Pediatrics and CF Center, Univ. of North Carolina, Chapel Hill, NC 27599.
Received 12 April 1998; accepted in final form 4 September 1998.
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