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An Old Fashion, Hi-Tech Answer
to Antibiotic Resistance: Bacteriophage Therapies Come Roaring
Back
It's the very cutting edge of biotechnology. And it's
the outgrowth of an 80 year-old discovery pursued most
aggressively in the former Soviet Union. The potential of
bacteriophages as anti-infectives has long been known, but has
also been nearly forgotten in modern times. Now the growing
problem of antibiotic resistance--and a growing understanding of
biology-- may return them to prominence.
By B.J. Spalding
Last year, during surgery for a life-threatening heart
disorder called Marfan syndrome, a Toronto woman suffered an
infection with a deadly strain of Staphylococcus aureus, one
resistant to all available antibiotics. As the woman's health
deteriorated, her family desperately appealed to Phage
Therapeutics International (Bothell, WA), a four-year-old
biopharmaceutical firm developing a bacteriophage that treats S.
aureus. A bacteriophage is a virus that typically kills just a
single strain of bacteria.
Since the product was in preclinical trials, the woman's
family had to get her doctor and her hospital to agree to allow
the use of the experimental therapy. They agreed, and during
subsequent surgery, physicians sprayed the phage directly onto
the woman's aorta and, following surgery, treated her with it
intravenously. Within 20 hours, all traces of the S. aureus
infection disappeared. Although the woman unfortunately died two
months later, it was her heart condition that killed her, not
her bacterial infection.
Richard Herman, Phage Therapeutic's director of microbiology,
expresses cautious optimism about this experience. "Since she
was just one patient, it's impossible to know if the phage cured
her infection or if something else did. All we can say for sure
is that the phage didn't harm her in any way," he says. But
there is reason for less guarded enthusiasm.
In the US, Phage Therapeutics--along with Exponential
Biotherapies (Port Washington, NY) and Intralytix (Baltimore,
MD)--leads the way in developing bacteriophages for therapeutic
uses. Phages are perhaps better known to the biotech community
as the engine of "phage display"--a means of generating
libraries of drug targets and of developing fully humanized
monoclonal antibodies, using the viruses as gene delivery
vectors. These applications are pursued by companies like Dyax
(Cambridge, MA), Morphosys (Munich, Germany), and Cambridge
Antibody Technology (Cambridgeshire, UK).
Yet the therapeutic application of phages, which were first
discovered over 80 years ago, far predate these newer uses.
Phages have a long history of use against bacterial infections
in Eastern Europe and the former Soviet Union. Moreover, in the
US in the 1930s, Eli Lilly (Indianapolis, IN) marketed about a
half dozen therapeutic phage liquids. In 1925, Sinclair Lewis
won the Pulitzer Prize for his novel, Arrowsmith, which depicted
a doctor who traveled the world using phages to treat his
patients, a character based on the real-life co-discoverer of
phages, Felix D'Herelle. But when antibiotics debuted in the
1940s, the once-celebrated phages slipped into oblivion, as
their biology was still poorly understood and, compared to
antibiotics, they were unreliable and hard to handle.
"Now that so many antibiotic-resistant strains of bacteria
have emerged, there might once again be a market for phages.
They may see a renaissance, a rebirth," states Carl Merril,
chief of the Laboratory of Biochemical Genetics at the National
Institute of Mental Health (NIMH, Bethesda, MD). And Torrey
Brown, Intralytix's chief executive officer (CEO), adds that
phages are the "natural enemy of bacteria. So it's our belief
that exploiting what would be the natural nemesis of a bacteria
of concern would be a far better strategy than creating a new
antibiotic that costs $40 zillion and that eventually has
resistance developed to it."
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Fighting the Resistance
Without a doubt, bacterial resistance to antibiotics has
become a huge problem. Bacteria have gained resistance to
antibiotics because of the gross overuse and misuse of these
drugs. Such use creates a situation--known as natural selection
in evolutionary terms--in which resistant bacteria can thrive in
an antibiotic's presence. This survival occurs largely through
random genetic mutations, which happen readily in bacteria and
sometimes produce a gene for a new resistance trait or one that
strengthens an already-existing resistance trait.
"The result is the development of 'superbugs.' Because
they're resistant to one antibiotic, they survive and, by
chance, mutate a gene that makes them resistant to another
antibiotic. This process goes on and on, until the superbug can
withstand not just a single antibiotic, but multitudes of
antibiotics," explains Alexander Sulakvelidze, an Intralytix
cofounder.
Already, many strains of four life-threatening bacteria,
including S. aureus, are completely resistant to every existing
antibiotic. Yet some strains of these four bacteria are still
susceptible to vancomycin, a generic antibiotic that many
public-health officials currently regard as society's "last line
of defense" against infectious disease.
The costs associated with bacterial resistance to antibiotics
are astronomical, particularly the human costs. Infectious
disease, in fact, has soared from the fifth-leading cause of
death in the US in 1980 to the third-leading killer today,
trailing only cardiovascular disease and cancer.
For these reasons, the antibiotic marketplace is headed for a
shakeup. Presently, worldwide antibiotic sales total roughly $25
billion a year. In the next few years, an anticipated $10
billion annually is expected to flow to new therapies targeting
today's antibiotic-resistant bacteria--whether these therapies
are new-generation antibiotics or alternative treatments, like
phages. These new drugs will in many cases steal market share
from older antibiotics.
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"Lunar-Lander Modules"
Though bacteriophage may seem exotic, at least when compared
to antibiotics, they're actually one of the most commonplace
organisms on earth, thriving wherever bacteria grow--in the
oceans, in our bodies, in sewage, and nearly everywhere else. A
milliliter of coastal seawater, for example, typically contains
a million phages, while in a milliliter of some fresh water
sources, like tap water, there may be a billion of them. In
fact, if all the phages on earth were gathered together and put
on a scale, they would outweigh the world's population of
elephants by a thousand fold or more.
About one-fortieth the size of most bacteria, phages have an
extraterrestrial appearance. "They look like lunar-lander
modules, with box-shaped heads stuffed with DNA, narrow and
rigid tails, and a tangle of spiderlike legs," states Richard
Carlton, the president of Exponential Biotherapies. Using their
legs to bind to specific receptors on specific strains of
bacteria, phages bore into the bacteria with their tails, and,
like living syringes, inject their DNA into the bacteria. The
phage DNA then forces the bacteria to produce copies of the
phage, on average about 200 phages in roughly 30 minutes, so
many that the bacterial cell walls burst, killing the bacteria
and releasing the phages.
Each of these 200 phages goes on to infect another bacteria,
each of which, after 30 minutes, bursts and releases another 200
phages. "So now, at just the second generation, you have 40,000
phages. At the third generation, you have 8 million phages, and
at the forth generation, you have 1.6 billion of them. And
that's for each single phage that you give to a patient," says
Exponential's Carlton.
"That's the tremendous potential of phage therapy," he
continues, "the exponential growth of the phage at the expense
of the bacterial host allows it to massively outstrip and
decimate the bacteria. Because, after all, while the phage is
replicating at a factor of 200, the bacteria is only replicating
at a factor of two--two and then four and then eight and then
16, etcetera." Phage Therapeutics's Herman adds, "Bacteriophage,
in contrast to antibiotics, don't diminish with time. They
increase over time. That's why they're so fast acting, why you
see an antibacterial effect within 12 to 24 hours. And that's
why you give fewer doses, only one to three administrations per
course of therapy."
Also, phages cause minimal side effects in comparison to
standard antibiotics. For one, phage-specific receptors aren't
present on mammalian cell membranes, so phages can't infect
mammalian cells. And even if a phage somehow got inside a
mammalian cell, mammalian restriction endonucleases, the enzymes
that chew up foreign DNA, mercilessly devour phage DNA.
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Exponential's Solid Foundation
Among the three companies targeting bacteriophages, it's
difficult to tell which of them is in the lead. What's crystal
clear, though, is that Exponential is by far and away the one
most willing to share information about itself. Perhaps that's
because Exponential--established in 1994 and currently carrying
10 employees--is the only one of the three companies with patent
protection solidly backing its platform technology.
Exponential's core technology involves numerous discoveries.
First, researchers wanted to eliminate bacterial toxins from
phage preparations, as both endotoxins and exotoxins are often
released into the culture medium when phages rupture bacteria
during replication. "These bacterial toxins can kill people just
as effectively as the bacteria can. They cause shock," states
NIMH's Merril, who oversaw the development of Exponential's
platform technology, though he currently has no ties to the
company. And, indeed, scientists were able to cut the toxin
content of phage preparations more than 100 fold by purifying
phages through cessium-chloride density centrifugation.
Yet researchers realized that what was most limiting the
efficacy of phage therapy was the rapid elimination from the
circulatory system of greater than 90% of the administered
phages by the reticuloendothelial system (RES), the body's
filtering organs, particularly the spleen. Assuming that RES
removal of phages depends primarily on the nature of phage
surface proteins, scientists developed a protocol to select for
phage variants that could evade RES capture. This "serial
passage" protocol involves, in this case, injecting mice with a
lambda phage, one specific for a strain of Escherichia coli that
causes blood infections, followed by isolation of the phage and
then regrowth of the phage in the E. coli strain. This cycling
of the phage is repeated 10 times.
Indeed, the cycling proved effective in selecting for a
lambda phage with an amino-acid mutation in a major head
protein, protein E. The mutation involved a change from glutamic
acid to lysine, a double-charge change from negatively charged
to positively charged. The mutation, furthermore, enabled the
phage to steadfastly avoid the RES. After one selection cycle,
for instance, serum phage levels dropped by four orders of
magnitude within 18 hours of injection, while after 10 cycles,
the drop was for less than one order of magnitude.
"For every 100,000 of the wild-type phages that we inject,
after 18 hours, there's only one left. So that's not a good
drug. It's very rapidly cleared. For the long-circulating
variants that we found, for every 100,000 that we inject, at the
end of 18 hours, there's 63,000 left. So you can see how much
clearance is slowed down. Instead of being taken out in two
minutes, when they can't reach the bacteria, the
long-circulating phages are taken out in the course of 24 hours.
And that makes all the difference in the world," explains
Exponential's Carlton.
To say that the long-circulating phages proved more
efficacious is an understatement. Mice injected with a lethal
dose of E. coli and the wild-type lambda phage got extremely
sick, before recovering from their blood infections. "They got
critically ill. It's like the equivalent of a human being in an
intensive-care unit," says Carlton. But mice injected with an
identical dose of E. coli, as well as the long-lasting variant
of the lambda phage, barely got sick at all. States Carlton, "It
takes a trained observer to see that they were even just a
little bit slowed down. In human terms, you feel like you have a
little bit of a cold."
With its core technology, Exponential has developed a
long-circulating phage that kills 95% of the strains of
vancomycin-resistant Enterococcus faecium that it has isolated
from patients throughout the US. The phage is currently
finishing preclinical trials, and Exponential expects to enter
it in clinical trials against blood infections within a few
months. "We've already heard from big pharma," says Carlton. "In
fact, they called us, one of the top five pharmas in the world.
We presented our animal data, and they were so impressed that
they want to go ahead and have a full-blown meeting in May."
Carlton adds, though, that for the "most part, I don't think
big pharma is really taking notice of phage research. But they
will when we have Phase II results, when we start saving lives.
They'll have to take notice then." Intralytix's Brown couldn't
agree more. "Once we've shown efficacy in Phase II trials," he
says, "big pharma will show interest. They're natural partners
as far as creating a distribution network and a supply system
and all those sorts of things. Their interest will peak when
they see that phages are safe and effective."
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Poland and the Soviet Union Never Quit
For his part, Carlton is a big believer in the clinical work
done on therapeutic phages in Eastern Europe and the former
Soviet Union. He cites a number of studies preformed relatively
recently by Stefan Slopek and his colleagues in Wroclaw, Poland.
In all, Slopek treated 550 patients with phage therapy, 518 of
whom had failed antibiotic therapy because of antibiotic
resistance. He administered phages either orally--after the
neutralization of stomach gastric acid--or topically against
infections caused by Staphylococcus, Salmonella, Klebsiella,
Escherichia, Proteus, and Psuedomonas.
Slopek, however, didn't include untreated patients as
controls in his studies. He defined positive results, moreover,
as improvement that was better than transient and that included
the healing of local lesions. "Response was gauged by clinical
evaluation. Clinical improvement alone was sufficient," says
Carlton, adding that although bacteriostasis--the inhibition of
bacterial growth--was achieved in the vast majority of patients,
it wasn't a necessary criterion of positive results.
Overall, Slopek reported such results in 508 of 550 patients,
or 92% of patients. These rates ranged from 75% in ulcerated
varicose veins to 100% in gastrointestinal infections,
pericarditis, and furunculosis, a skin infection. In one study
of 138 patients that looked at side effects, furthermore, Slopek
found that only three patients suffered adverse reactions, with
oral administration of phages leading to gastrointestinal
troubles and topical application causing allergic symptoms.
Carlton also cites a study performed a while back by a pair
of physicians, Sakandelidze and Meipariaini, on 236 patients in
Tbilisi, Georgia, formerly a part of the Soviet Union. The
patients had antibiotic-resistant infections--including
osteomyelitis, lung abscesses, and postsurgical wounds--that
were populated by Stapylococcus, Proteus, or Streptococcus. They
were treated with pyophage, a mixture of phages active against
all three bacteria, or with diphage, a phage mixture active
against two of the three. The mixtures were applied
subcutaneously or through surgical drains for five to 10 days,
and in 92% of the patients, the mixtures eliminated bacterial
growth.
Yet Carlton cautions that, as with the Polish studies, the
"efficacy of the phage in treating these infections was
determined by qualitative clinical observation. Also, the
methods for preparing the phage weren't described." What's
obvious, though, is that the Soviet and Polish scientists have
overcome many of the problems that plagued early phage research
in the US. Among these problems were difficulty in selecting
phages specific for targeted bacteria, failure to purify
bacterial toxins from phage preparations, and failure to
neutralize stomach acid before oral administration of phages,
among a host of other troubles.
Carlton concludes that US researchers should try to replicate
the Polish and Soviet studies under placebo-controlled,
double-blinded conditions. "That way, Western scientists could
learn the details of how Russian and Polish researchers prepare,
isolate, and administer phage," says Carlton. Elizabeth Kutter a
professor of biochemistry at Evergreen State College (Olympia,
WA) agrees, stating, "We need to draw as much as possible on the
largely unknown body of knowledge that has accumulated in Poland
and many parts of the former Soviet Union as we again explore
phage therapy. We also need to give credit where it's due for
the many years of hard, careful work that their scientists have
invested in the field."
Like Carlton, Intralytix's Brown is a huge supporter of the
former Soviet Union's clinical work with therapeutic phages. So
much so that many of his two-year-old company's roughly 20
employees either used to work in Tbilisi at the world-renowned
Eliava Institute, or still do work there, including Intralytix's
cofounder, Sulakvelidze. "We formed Intralytix to take advantage
of the knowledge about phage therapies that already exists over
there," Brown says. "What we'll do is spend the time, effort,
and do the studies needed to get those therapies approved in the
US. Obviously, most people believe the therapies are safe and
effective. They've been using them for decades."
In the heyday of the Soviet use of phage therapeutics, the
Eliava Institute was at the hub of the country's wide network of
phage production and distribution. Indeed, just before the
Soviet Union's break up, phage preparation was carried out on an
industrial scale, employing 1,200 people. Tons of tablets,
liquid preparations, and spray containers of carefully selected
mixtures of phages for therapy and prophylaxis were shipped
throughout the country each day.
These mixtures were generally available both over the counter
and through physicians, with the largest use in hospitals, to
treat both community-acquired and hospital-acquired infections,
either alone or in tandem with antibiotics. "Phages played a
particularly important role when antibiotic-resistant bacteria
were found," says Evergreen State's Kutter. At the time the
Eliava Institute was home to the largest phage library in the
world, as it housed a collection of over 300 phage clones.
Yet today, after years of bloody civil war following the
Soviet Union's collapse and Georgia's gaining of independence,
the republic is in free fall, rapidly drifting toward national
poverty. The Eliava Institute, too, nearly collapsed, saved only
by the determination of its individual researchers, who worked
for long stretches without pay, and by sporadic funding efforts
from the West, such as the initiative by Intralytix. Perhaps
most telling, says Evergreen State's Kuttter, are the old
fermentation vats that used to churn out phage preparations for
use throughout the Soviet Union, as they now stand abandoned in
a room with shattered glass in its window frames. Also, because
electricity runs for only part of the day and because phage
cultures must be refrigerated, Eliava has lost about half of its
phage library.
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Intralytix and Phage Therapeutics
For its part, Intralytix is initially focusing on technology
developed by Eliava for the use of phages for prophylactic
purposes. The company is currently working with a multinational
food processor on the problem of "environmental decontamination"
and is initially targeting such common pathogens as Salmonella,
Campylobacter, Listeria, and E. coli H7:0157, states
Intralytix's Brown. "With foods that are contaminated by the
environment, you might decontaminate the environment, and then
you might, on the other hand, decontaminate the food before it's
finally sold," he says.
"Preventing the bacteriologic problems that get to humans is
the step before you would need to treat humans," says Brown.
"That's what we're doing with food processors, as opposed to
what we'd do with big pharma. All our contacts with big pharma
are preliminary anyway."
Phage Therapeutics, for its part, is working on a phage that
is lethal to strains of S. aureus and S. epidermidis that are
resistant to the antibiotic methicillin. It has, in fact,
developed a particular phage that kills 93% of a broad spectrum
of over 1,000 of these strains that were isolated from patients
in the US, Canada, and South America. The phage is presently is
preclinical studies, and Phage Therapeutics plans to enter it in
clinical trials against eye infections within a year. The
company, however, hasn't had any contact with any pharmaceutical
firms regarding the product. "I haven't spoken to anyone from
big pharma about phages," says Phage Therapeutics's Herman.
Yet something about Phage Therapeutics--a six-employee firm
that lost a little over $3 million between December of 1996 and
April of 1999--simply doesn't seem to settle quite right.
Earlier this month, Caisey Harlingten--the company's chairman,
director, and original founder--resigned. In January, Richard
Honour quit, the firm's president and CEO, as did its chief
financial officer, Wayne Rebich. These two executives had only
come aboard six months earlier, in June of 1999. Also in
January, Weems & Co., the investment banker for Phage
Therapeutcs, backed out of a deal to raise $6 million in equity
financing for the company. "I don't know why it's all happening
around the same time. I don't know if all of these events are
related or not," says Herman.
How the Food and Drug Administration (FDA, Rockville, MD)
will react toward phages is uncertain, of course. "The FDA is
well aware of the problem of antibiotic-resistant organisms, and
it recognizes the critical need to come up with ways to treat
these organisms. So it would probably be sympathetic to new
methods, or even old methods, which is what phages are," states
NIMH's Merril. And Herman adds, "So far it doesn't seem that the
FDA is going to dismiss phages out of hand. Rather, it seems
interested to see if they can be developed."
What's also uncertain is whether bacteria can develop
resistance to phages, as they have to antibiotics. Says Merril,
"As bacteria become resistant to phages, phages will evolve to
get around that resistance. There's a constant battle going on
between the two.But the exponential growth of phages will
decrease the chance of resistance appearing in bacteria."
Intralytix's Sulakvelidze states, "It's a biological arm's race.
Super bacteria will develop resistance against existing phages.
But then you'll find super phages evolving against the super
bacteria. It's a never-ending process. That's its beauty." And
Intralytix's Brown adds, "The bacteria doesn't want to be
susceptible to the phage. But the phage wants to have the
bacteria susceptible, so it has something to eat and a place to
multiply. So the belief is that the phage will evolve along with
the bacteria that it targets."
But the greatest uncertainty is what the future holds for
phages. "I see a lot of potential," says Merril. "But I also see
phage development as not a simple task. It's one in which
companies will have to invest a lot of time, money, and
man-hours. Companies shouldn't expect phages to make money
overnight, nor should investors."
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