Discussion
The primary goal of the study was to assess the potential of the oral
intestine-specific NHE3 inhibitor tenapanor to prevent intestinal
obstruction in CFTR null mice. Several major obstacles were noticed
during the study preparation and had to be addressed: Tenapanor quickly
precipitates from aqueous solutions at neutral or alkaline pH. Since
microencapsulated tenapanor for use in mice is not available, it had to
be applied by gavage, and dose finding studies were necessary. We aimed
at finding a tenapanor dose and application frequency that resulted in a
stable but mild increase in stool water, because diarrhea is quickly
lethal for mice (Barone et al., 2009). 30 mgkg-1tenapanor, applied twice daily by gavage, resulted in a mild increase in
stool water, and it did not significantly influence the body weight of
the mice. Surprisingly, the required dose was the same inCftr -/- and Cftr -/-mice, suggesting relative independence of the CFTR mediated route of
fluid secretion and the NHE3 mediated route of fluid absorption. It is
likely that the microencapsulation of tenapanor available for use in
humans will permit lower doses in the CF population. An optimal dosing
regimen to prevent obstructive episodes in CF patients without causing
diarrhea or other unwanted side effects needs to be established.
The Cftr -/- mice experienced a mild loss of
body weight in both the tenapanor and vehicle group, which may be
related to an increased stress level due to the twice daily gavage. The
lack of effect of tenapanor on body weight is an important finding,
because it is known that NHE3 function is required for PEPT1-mediated
dipeptide absorption (Thwaites & Anderson, 2007). It is in agreement
with a lack of reduction of PEPT1-mediated substrate absorption during
short term tenapanor ingestion in healthy volunteers (Johansson et al.,
2017). However, NHE3 has also been shown to be involved in the
absorption of amino acids (Anderson & Thwaites, 2005) and
micronutrients (Shawki et al., 2016). Therefore, careful observation is
necessary during long term studies in CF patients.
Replacing the drinking fluid containing the osmotic laxative with tap
water may result in an initiation of an obstructive episode within the
first day (which becomes evident only several days later); we therefore
started the tenapanor gavage on the last day of the oral laxative
administration. It is well known that the incidence of obstructive
episodes (always lethal) in Cftr -/- mice is
very high during weaning (Snouwaert et al., 1992), and 90%Cftr -/- mice are dead by day 30 unless
preventive measures are taken (Clarke, Gawenis, Franklin & Harline,
1996), and decreases in adulthood. Previous studies that aimed at
pharmacological prevention of obstructive episodes inCftr -/- mice have therefore administered the
pharmaceutical agent to mice aged 3-6 weeks (Walker, Simpson, Levitt,
Boyle & Clarke, 2006), and 20 days, respectively (Lord et al., 2018).
For our study, the mice had to tolerate twice daily gavage and
preferably not loose body weight due to this procedure; we therefore
chose the age range of 9-13 weeks. This explains why the percentage of
obstructions is lower in our study compared to the studies mentioned
above.
The primary goal of our study was the questions whether intestinal
obstructive episodes can be prevented by tenapanor in a test group at
high risk for obstructions, which is the CFTR null mouse (no CFTR
protein present). Although our study was conducted in an age group in
which the mice have a decreased risk compared to younger age, the
results showed that the risk was still high (46% within the observation
time) and was reduced to 8% with tenapanor treatment.
Apart from obtaining a positive result of the study, we wanted to
understand how tenapanor conveyed this reduction in obstructive episodes
at a cellular and molecular level. As previously studied in intestinally
perfused mice, the direct effect of tenapanor will be a reduction of
NHE3-mediated sodium and fluid absorption and an increase in the luminal
alkalinity in both the small and large intestine (Tan et al., 2021),
which was also seen in the stool of the mice in this study (Figure 1g).
A normalization of the delayed GTT in the tenapanor-treatedCftr -/- mice is likely another key effect
protecting the mice against obstructions.
Somewhat surprisingly, tenapanor treatment resulted in the reversal of
other features of the intestinal abnormalities inCftr -/- mice. Of particular interest to us was
the finding that tenapanor treatment reversed the cryptal
hyperproliferation in the ileum and proximal colon. A hyperproliferative
response has been observed previously inCftr -/- small (Gallagher & Gottlieb, 2001) and
large intestine (Tan et al., 2021). The intracellular pH ofCftr -/- deficient enterocytes is more alkaline
than that of corresponding Cftr +/+ cells
(Simpson, Gawenis, Walker, Boyle & Clarke, 2005). This feature is
preserved in the crypts including the stem cells of intestinal organoids
(Liu, Walker, Cook, Ootani & Clarke, 2012) and is causally related to
the increased proliferative rate, which is preserved inCftr -/- intestinal organoids (Strubberg et al.,
2018). The presence of NHE3 has been reported both in undifferentiated
and differentiated intestinal organoids (Foulke-Abel et al., 2016).
Immunohistochemical staining of NHE3 overlaps with that of CFTR in the
villus region and cryptal mouth region (Jakab, Collaco & Ameen, 2011).
Therefore, it is possible that chronic tenapanor application reverses in
part some of the pathological effects of the high intracellular pH on
proliferative activity. A similar observation was made by Bradfordet al . in their studies of mice which were homozygous negative
for CFTR, for NHE3, or for both genes. While bothCftr -/- and Nhe3 -/-mice displayed an increased cell proliferation in duodenal crypts, this
increase was significantly reduced in the crypts ofCftr-/-/Nhe3-/- mice
(Bradford, Sartor, Gawenis, Clarke & Shull, 2009). Thus, long term
tenapanor treatment, if proven safe in CF patients, may even have to
potential to lower the risk for GI malignancy.
Another striking observation in tenapanor treated mice was a reduction
in intestinal mucus accumulation. We previously reported an increase in
the goblet cell counts, as well as enlarged goblet cell theca, in the
mid-distal colon of Cftr -/- mice that did not
display evidence of inflammation (Kini et al., 2020; Tan et al., 2021).
Because of the well documented crypt-villus hyperproliferation inCftr -/- intestine (Gallagher & Gottlieb, 2001;
Tan et al., 2021), we used a fluorometric method of quantifying the Muc2
immunofluorescence in relation to that of the Dapi stained nuclei in
this project. The total as well as the extracellular Muc2/Dapi
immunofluorescence was significantly reduced in tenapanor vs
vehicle-treated mice in the ileum and proximal colon, while it was very
high when an obstruction was present.
We also noted a shift in the expression of the Muc genes, with a
non-significantly lower expression of Muc2 mRNA and a
significantly higher Muc1 mRNA. The 21 day treatment with
tenapanor reduced Muc1 mRNA expression compared to vehicle
treatment. The somewhat lower Muc2 mRNA expression was also seen
in the present study, with no effect of tenapanor. It has been shown
that Muc1 deletion, although not a major component of intestinal
mucin, reduces the thickness of the firmly adherent mucus layer
(Malmberg et al., 2006). Muc1 deleted CF mice have reduced amounts of
intestinal mucus and a better survival compared to Muc1-expressing CF
mice (Parmley & Gendler, 1998). It is therefore likely that the
observed tenapanor induced changes in Muc1 expression and mucus
accumulation play a major role in reducing obstructive episodes.
The intestinal tract of patients with cystic fibrosis display an
inflammatory phenotype in the absence of bacterial infection or enzyme
induced fibrosing colitis. Multiple explanations have been provided,
including bacterial overgrowth due to dysmotility, oxidative stress,
intracellular accumulation of mutated CFTR protein, and lack of
pancreatic and/or intestinal defensins (Bruzzese et al., 2004; Crites et
al., 2015; Galli et al., 2012; Lisowska, Mdry, Pogorzelski, Szydłowski,
Radzikowski & Walkowiak, 2010; Raia et al., 2000; Werlin et al., 2010).
In mice, chronic exposure to PEG containing laxative in the drinking
fluid both increased survival and reduced the inflammatory signature in
the intestine of these mice (Bradford, Sartor, Gawenis, Clarke & Shull,
2009; Clarke, Gawenis, Franklin & Harline, 1996). We had previously
reported no difference in proinflammatory cytokines in the different
segments of Cftr -/- andCftr +/+ littermates while on the PEG containing
laxative, yet several features that had been correlated with an absent
CFTR function (in addition to the reduced anion and fluid secretory
response) were present: increased enterocyte pHi, an
increased mucus content in the goblet cell theca, crypt-villus
elongation, decreased surface pH with fluid hyper-absorption (Kini et
al., 2020; Tan et al., 2021). In this study, a discreet but significant
increase in the number of mast cells was observed in the lamina propria
of the intestinal tract of the vehicle treatedCftr -/- mice compared toCftr +/+ mice, which was completely or partially
prevented by tenapanor treatment. A very discreet increase in neutrophil
infiltration was seen in Cftr -/- ileum and
mid-distal colon, not accompanied by an increase in any of the
proinflammatory cytokines. In contrast, a strong increase in neutrophil
infiltration and in the expression of proinflammatory cytokines was only
seen in mice with an obstruction, and was restricted to the intestinal
segments proximal to the obstruction that were filled with fecal matter.
These segments also exhibited severe alterations in the mucosal
architecture. Thus, the intestinal inflammation was a sequelae of the
obstruction, and not its cause.
A relative shortcoming in our study is that the application mode of the
drug is relatively invasive, and therefore the study duration is fairly
short. The number of mice permitted for the study by the animal
committee was sufficient to reach the primary endpoint, but was not
enough to determine significance for the reversal of every studied
CF-associated intestinal abnormalities by tenapanor treatment in every
intestinal segment. When we performed the GTT experiments, we noticed
that the stool water content was still elevated in theCftr +/+ mice 11 hours after the last dosing,
but not in the Cftr -/- mice, suggesting that
more frequent dosing, not feasible with this form of application, may
have been optimal. Obviously, this problem will not exist in CF patient
trials. For a tenapanor trial in CF patients, it will be necessary to
define the patient population at high risk for intestinal complications,
and preferably test its prophylactic potential. Patients with meconium
ileus at birth and prior DIOS episodes, and lung or liver transplanted
patients are at particularly high risk, and CF diabetes, pancreatic
insufficiency and female gender also carry a higher risk (Houwen et al.,
2010; Lavie et al., 2015; Munck et al., 2016). While corrector and
potentiator therapy is beneficial for CFTR-dependent functional outcome
parameters (Graeber et al., 2022; Ooi et al., 2018), adjunctive therapy
specifically targeting the constipation and obstructive episodes appears
necessary in many CF patient subgroups.
Tenapanor treatment is considered safe for humans (Block et al., 2021;
Sinagra et al., 2020). It has not been tested in children so far, and
the longest observation time in a published trial is 52 weeks. Given the
good safety profile, clinical trials testing the effect of tenapanor on
the intestinal symptoms of CF patients seem warranted.