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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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