A drug that blocks the intestinal pathogen without
killing resident, beneficial microbes may prove
superior to antibiotics, currently the front-line
treatment for the infection.
Stanford University School of Medicine scientists
successfully defeated a dangerous intestinal
pathogen, Clostridium difficile, with a drug
targeting its toxins rather than its life.
By not aiming to kill the pathogen with antibiotics,
scientists were able to avoid wiping out sizeable
numbers of beneficial gut microbes. And while their
study was performed in mice, the drug used has
already been tested in clinical trials to treat
other, unrelated conditions. So the researchers
believe it could be moved rapidly into human trials
for the treatment of C. difficile, as well.
The findings, published online Sept. 23 in Science
Translational Medicine, constitute the first-ever
demonstration of a small molecule’s ability to
disarm C. difficile without incurring the collateral
damage caused by antibiotics.
C. difficile is responsible for more than 250,000
hospitalizations and 15,000 deaths per year in the
United States, costing the country more than $4
billion in health-care expenses, said the study’s
senior author, Matthew Bogyo, PhD, professor of
pathology and of microbiology and immunology. Lead
authorship of the study is shared by Kristina
Bender, PhD, a former postdoctoral scholar in
Bogyo’s lab, and Megan Garland, a student in the
Medical Scientist Training Program.
“Unlike antibiotics — which are both the front-line
treatment for C. difficile infection and,
paradoxically, possibly its chief cause — the drug
didn’t kill the bacteria,” Bogyo said. Instead, it
disabled a toxin C. difficile produces, preventing
intestinal damage and inflammation and allowing the
gut to be repopulated by healthy bacteria that had
been decimated by earlier rounds of antibiotic
treatment, as well as by C. difficile-induced
intestinal changes.
About one in 20 people, and possibly many more,
harbor C. difficile in their gut, said study
co-author Justin Sonnenburg, PhD, professor of
microbiology and immunology, who has conducted
pioneering research on the trillions of microbes
constituting our intestinal ecosystems. Usually, the
pathogen causes no harm, he said. But in those with
immune systems weakened by age, chemotherapy or
antibiotics that wipe out their “lawn” of beneficial
intestinal microbes, C. difficile can get a foothold
and cause changes that damage the gut.
Plus, the pathogen can dehydrate and condense into
shrunken, long-lived spores, making it difficult to
get rid of. Most C. difficile infections originate
in settings such as hospitals, clinics and
assisted-living facilities.
Making matters worse, in a quarter of patients who
get it, the infection recurs despite antibiotic
treatment. When it does, antibiotics succeed in
eliminating it only 25 percent of the time. About 7
percent of infected people die within 30 days of
diagnosis.
Treatments for C. difficile infection include fecal
transplants, which are often effective. But this
treatment’s long-term safety is difficult to
ascertain, as a stool sample from any given donor
contains its own mix of intestinal microbes, and
some could have adverse effects on a recipient’s
health. “We don’t have the tools to be able to
screen for everything in a donor’s stool,” said
Sonnenburg, noting that gut bacteria have been
implicated in obesity, as well as in neurological
changes.
Bogyo’s group has extensive expertise in studying
the activity of proteases, proteins capable of
slicing up other proteins. A few years ago,
co-author Aimee Shen, PhD, a postdoctoral scholar in
Bogyo’s lab who is now an assistant professor at the
University of Vermont, found that C. difficile’s
main toxins — secreted proteins known as Toxin A and
Toxin B — contain nearly identical sections with
protease activity.
Moreover, she found, once the toxins are taken up by
cells lining the mammalian gut, these sections
become activated, setting in motion a chain of
intracellular events that causes intestinal
inflammation and tissue damage.
This is a positive development for C. difficile,
which thrives in the new environment it has created.
But it’s another story for myriad other bacterial
species residing in the intestine — and disastrous
for the infected individual’s health, with symptoms
ranging from severe diarrhea to intestinal lesions
to death.
Bogyo’s team has developed ways of conducting
high-throughput screens of small molecules to
speedily test their ability to inhibit or enhance
the activity of proteases. They put this technique
to work in search of small molecules that
specifically blocked the C. difficile toxins’
protease activity.
“We figured that a molecule that interfered with the
pathogen’s virulence could prevent inflammation and
the disruption of colon tissue without making the
intestinal environment inhospitable to normal,
beneficial bacteria the way antibiotics do,” said
Bogyo. That would lay the groundwork for the “good
guys” to make a comeback.
In the first of a series of experiments, the
investigators separately incubated each of 120,000
different small molecules with the
protease-containing piece of C. difficile’s primary
toxin, Toxin B. Then, they added a toxin-activating
factor and, using a test they’d devised in which
protease activity is signaled by fluorescence,
looked for drugs that shut down that activity. They
identified hundreds of such substances, including a
number of compounds with known biological activity.
Bogyo and his associates focused on a compound
called ebselen because, in addition to having a
strong inhibitory effect, ebselen also has been
tested in clinical trials for chemotherapy-related
hearing loss and for stroke. Preclinical testing
provided evidence that ebselen is safe and
tolerable, and it has shown no significant adverse
effects in ensuing clinical trials.
Bogyo’s team conducted another test to see how
ebselen affected human cells. The team incubated the
complete Toxin B molecule with the cells in the
presence or absence of ebselen. When Toxin B was
activated inside a cell, it induced internal damage
that caused the cells to assume a rounded shape and
die. Ebselen prevented that from happening.
Realizing they might have a potentially effective
drug on their hands, Bogyo and his colleagues
brought in Sonnenburg, whose lab is adept at using
mouse models of C. difficile infection.
The researchers incubated Toxin B in a solution
either containing or lacking ebselen and injected it
into mice, then monitored the animals for three
days. All of the mice injected with ebselen-pretreated
toxin survived, while all of the mice that received
the untreated toxin were dead within 48 hours.
In a final set of studies, Bogyo and colleagues
tested ebselen in a mouse model that more accurately
mimics a clinical scenario in which high-risk
individuals are treated prophylactically or at the
first symptoms of recurrence.
After rounds of multiple antibiotics, the
researchers introduced a virulent,
multi-drug-resistant C. difficile strain and then
began oral dosing with ebselen. They observed a
nearly complete block of inflammation and damage to
colon tissue as the result of ebselen treatment.
The upshot of this and other experiments conducted
by Bogyo’s team is that using ebselen to disable a
toxin in C. difficile was enough to significantly
reduce the clinical symptoms of the infection and
block the persistent gut damage in mice.
Bogyo said he hopes to move the drug rapidly into
clinical trials for treating C. difficile infection.
Other Stanford-affiliated co-authors of the study
are postdoctoral scholars Andrew Hryckowian, PhD,
Matthew Child, PhD, and Ehud Segal, PhD; former
graduate students Jessica Ferreyra, PhD, and Aaron
Puri, PhD; life science research professional Steven
Higginbottom; David Solow-Cordero, PhD, director of
the High Throughput Bioscience Center in the
Department of Chemical and Systems Biology; and Niaz
Banaei, MD, associate professor of pathology and of
infectious diseases.
For more information
A small-molecule antivirulence agent for treating
Clostridium difficile infection
Science Translational Medicine 23 Sep 2015:
Vol. 7, Issue 306, pp. 306ra148
DOI: 10.1126/scitranslmed.aac9103
Stanford University School of Medicine
MDN |