Biotech Values

[DewDiligence]

Frogs, sharks, and Michael Zasloff

[By special request from ‘mtforeman’ on the Yahoo board... The December, 2000 article from now-defunct Red Herring magazine about GENR founder, Dr. Michael Zasloff. Near the end of the article is a famous tidbit about Trodulamine, oops, I mean Trodusquemine, which I won’t spoil by revealing it here.

This article is a fine read for historical purposes and for sheer amusement, but I do not believe Trodusquemine is going anywhere in the near future. I would be overjoyed to be wrong about this, of course.]

http://www.redherring.com/Article.aspx?a=6587

>>
Nature never lies, but you have to ask the right questions.

December 4, 2000
By Thomas Maeder

Michael Zasloff was not even thinking about creating a drug when he discovered one, launching a new field and a new company and turning his own life upside down. He was doing basic research on the mechanism of RNA transport at the National Institutes of Health , where he was the highly respected chief of the human genetics branch of the National Institute of Child Health and Human Development.

Dr. Zasloff, then 40, extracted nuclear material from the huge eggs of the African clawed frog, Xenopus laevis. Rather than killing the frogs and discarding their carcasses, as most scientists do, he removed the eggs through two small incisions, sutured the wounds, and let the animals convalesce for two weeks before sneaking them down the elevator in writhing orange biohazard bags and releasing them under cover of darkness into a branch of Rock Creek, their days of government service over.

In the spring of 1986, Dr. Zasloff noticed a baffling phenomenon. His tanks were filthy, teeming with microorganisms, yet the frogs' wounds rarely became infected or inflamed. If their immune systems had been protecting them, there would have to have been inflammation. What else could protect them? When Dr. Zasloff asked frog experts or immunologists for an explanation, they dismissed him with a disinterested shrug. "Maybe they're too stupid to get sick," one suggested.

The frogs' curious robustness was all the more puzzling to Dr. Zasloff, a physician as well as a basic scientist, because he knew of a man who at that very moment lay close to death due to a water-borne infection, despite the best treatment that doctors could provide. What did the frog have that medicine could not provide?

Dr. Zasloff collects medical anomalies. Occasionally he hauls one out and tries to fit a new bit of knowledge into an old puzzle whose missing parts have burned their silhouettes into his brain. Why do the intestines of people with Crohn's disease show skip lesions, alternating bands of healthy and diseased tissue? Why does excessive bone growth accompany some pulmonary diseases? Why does rheumatoid arthritis progress symmetrically? And the question that had plagued him since his residency in pediatrics at Boston Children's Hospital, why are the lungs of children with cystic fibrosis colonized by bacteria soon after birth? Or rather, why aren't everyone's lungs similarly infected? Every breath sucks legions of microorganisms onto the warm, moist, vulnerable tissue of our lungs, yet for most of us, there is no immune response: our lungs are not chronically inflamed. Dr. Zasloff had hypothesized that some local antimicrobial must protect the body's moist surfaces, but he had never known how to study it. Now, looking at the frog, he did.

What the frog possessed was surprisingly simple and amazingly powerful. Working alone, telling no one, haunted by the fear that he might be wrong, Dr. Zasloff isolated, purified, and characterized the frog's defensive molecule, found and sequenced its gene, and determined its remarkable spectrum of activity. It was a 23-amino acid, straight-chain peptide that had no particular structure in water. But when one of these molecules, which Dr. Zasloff called magainins, from the Hebrew word for "shield," came into contact with certain cell membranes, it snapped into a helical coil that floated half-submerged in the surface. At a high enough concentration, these coils aggregated to form pores, disrupting the membrane and bursting the cell.

Detergents or toxins like melittin, the active component of bee venom, also disrupt cell membranes, but do so indiscriminately. Magainins breach only the membranes of microorganisms, including many major pathogens, leaving those of mammalian cells unscathed. Bacteria are adept at evolving resistance to most antibiotics by changing their targets, snipping them apart, or keeping them out of a cell, but resistance to magainins would demand a virtually impossible alteration of the bacterial membrane itself. Bacteria don't develop resistance to magainins; indeed, microbiologists have failed in attempts to breed resistance into them.

In his first paper on his discovery, published in Proceedings of the National Academy of Sciences, Dr. Zasloff boldly inferred that magainins were not unique to frogs, but that similar molecules -- of great promise in the fight against pathogens -- would be found in the gut, mouth, and respiratory tract of human beings. He also speculated that a defect in the system might be implicated in cystic fibrosis, which has subsequently proved to be true -- the defect lies in the excessive salt in CF patients' lungs.

The response was extraordinary. "Frog's Skin Yields Powerful Antibiotic," blared a headline on the front page of the Washington Post. A New York Times editorial compared Dr. Zasloff to all three discoverers of penicillin rolled into one. The national and international print and broadcast media picked up the charming story of "Dr. Kermit" and his "ribbetting evidence," and Dr. Zasloff was besieged with job offers from academia and licensing inquiries from drug companies.

Dr. Zasloff's first impulse was to donate magainin to the American people, whose taxes had funded his NIH work. He naively approached the U.S. Food and Drug Administration for help but was told derisively that the FDA exists to regulate drugs, not to make them. The NIH offered little help. Eventually, people more worldly in business than he persuaded him that only a private company would have the capital and motivation to create a new drug, and could bring in the skills needed for the process. But scientists and business people don't always see eye to eye, as Dr. Zasloff would discover.

"Michael is a very smart fellow," says Barry Berkowitz, a former SmithKline Beckman executive hired in 1988 to run a new company called Magainin Pharmaceuticals , founded by HealthCare Ventures, which licensed Dr. Zasloff's patent from the government. "But of course, the world is full of very smart fellows. For Michael to have tried to turn his molecule into a drug on his own would have required about five major miracles in a row. He would have been better off joining a religious order than trying it on his own."

Magainin Pharmaceuticals was established just north of Philadelphia and began the tasks of raising capital, building a facility, identifying clinical applications with good market potential, and screening synthetic magainin analogs for better or different therapeutic profiles. A slightly altered version of the original frog molecule became the company's lead compound, MSI-78.

Dr. Zasloff resigned from the NIH -- a painful separation, but the prerequisite for any company involvement -- and accepted joint appointments at Children's Hospital of Philadelphia and the University of Pennsylvania, where, with a score of graduate students and postdoctoral assistants, he would study the basic science of host defenses while putting in some time as a company scientific consultant. He delighted in a company founded on his ideas and reveled in perks like his company Jaguar. Yet he complained of bureaucracy and plodding procedures and could not adapt to the corporate mold. Years later, on a road show as the company was preparing to go public, he rattled on to investors about how he cared nothing about money and only yearned to see magainins become a drug that cured people of disease. Dr. Zasloff says Jay Moorin, the company's second CEO, drew him aside and chastised him severely.

"'Don't ever tell investment people that you don't care about money,' he told me," Dr. Zasloff remembers. "'Whether you care about it or not, they do. It's their life. It's what they're about. If you say you don't care about it, you sound insane, or like a creature from another planet. They won't understand you, and they certainly won't trust you, because you don't live by their rules.'" Good advice, but hard to follow for a man who had never even owned stock before, a man whose heart remained in the clinic and the lab.

Wherever Zasloff worked, he could never stop being a hands-on scientist. He had always been noted for his "brain/hand coordination" -- a deft knack for transforming a scientific question into a rapid, simple, effective experiment -- and for his intense, contagious enthusiasm. "If this is true, a whole new field of science has opened, here and now, before your eyes," he grandly announced at a lab meeting, referring to the results of a postdoctoral assistant's experiment the week before. He paused while the import of his statement sank in. Then a flicker of doubt crossed his face. "And if it's not true," he chuckled, "then nothing has happened." He paused again and his eyes lit up. "But whether it's true or not, this is what makes you want to do science."

"Mike can drive you nuts," says Karen Moore, who collaborated on one of the lab's major discoveries. "I'm maybe 40 percent sure of something on Monday, then 30 percent on Tuesday, and up and down as the evidence goes. Mike is always 100 percent certain...until the next day, when he's abandoned it. 'Oh, that's wrong. It's gone. But I have a new idea!' and he's 100 percent certain of that. It can make you insane. But otherwise, it's a wonderful way to live."

When I first met Dr. Zasloff in 1989, he had set his lab the grand challenge of mapping the whole uncharted domain of host defenses. He saw magainins as merely the tip of an elaborate system. Understanding their biology, where they were produced, how they were packaged and released, and how they fit in with the other defenses would lead to other, better antibiotics and perhaps teach us to manipulate the innate systems that control their expression.

But when Dr. Zasloff looked inside other amphibians, seeking to define the whole set of antimicrobial peptides, he was at once frustrated and excited to discover that every frog and toad had its own molecule, no two of which were the same. Nature had cleverly created the same selectively destructive helical structure out of widely varying amino acid sequences, and thus given bacteria less of a chance to evolve resistance.

He searched in other animals and organs as well -- fruit flies, chickens, salmon, pig eyes and fallopian tubes, cow windpipes, rat stomachs, mouse lungs, and human placenta. People who read about magainin wrote to suggest their own favorite disease-resistant creatures -- starfish, earthworms, sea urchins, Moses sole, whales, codfish, alligators. Some claimed that certain melons or the herb epazotй resisted decay, or that compost helped heal a gardener's scrapes. Dr. Zasloff had given them, as himself, a conceptual template for discovery.

One nephrologist extolled the incredible hardiness of dogfish sharks, which he used for kidney research. Dr. Zasloff was not particularly impressed -- it began to seem as though everything was hardy -- though he was struck by a strange feature in the creature's anatomy. The dogfish shark has the longest gestation period of any creature, two full years, and during that time the fetal pups float in a uterus that is open to the dirty sea, unprotected by the mother's immune system, which is not much good anyway. "If a human being had the immune system of a shark," Dr. Zasloff liked to say, "he would be dead."

Dr. Zasloff imagined that the shark might hold clues to a systemic equivalent of magainins, something that protected the inside of the body much as magainins shielded its surfaces.

The shark antimicrobial turned out not to be a peptide, as Dr. Zasloff confidently assumed, but an aminosterol, a cholesterol-like molecule, whose chemical properties eluded his peptide-separation techniques for many months. He marveled that nature had engineered a totally different solution to the host defense problem, and thought that "squalamine," as he called it, was far more powerful and important than his original discovery. "Magainin is a wimp," was a highlighted quote in a New York Times interview about squalamine, to the embarrassment of the company dependent on magainins' commercialization.

In 1993, soon after Magainin Pharmaceuticals went public, Dr. Zasloff got into a dispute with Children's Hospital, which threatened to cut his lab space in half because his million-dollar-a-year private grants did not include enough overhead for the administration's liking. He resigned his academic posts. To his surprise, Magainin did not hold out its arms in welcome; as is often the case, the biotech startup wanted nothing from its scientific founder other than his initial idea. The company reluctantly gave him a small bench space in the general peptide lab, with little funding for equipment or supplies. "That's all right," he said. "I'm working like Pasteur. This is the way that real science is done." And he managed to do great science with nothing.

On the evening before Thanksgiving 1993, I joined Dr. Zasloff on the loading dock at Magainin Pharmaceuticals. He had found an inexhaustible supply of shark livers, the organ richest in squalamine, from a fishery in New Hampshire that was happy to dispose of its waste. Now he had to process hundreds of pounds of tissue to obtain the grams of squalamine he needed for research, and because his corporate colleagues -- focused on taking magainin to market -- would not spare him the resources, he did the job on his own, using the simplest tools: a meat grinder, some giant stainless steel stock pots, a 3-foot-long plastic paddle, a bilge pump, and a set of 55-gallon garbage cans. His CEO had banished him to the dock, after closing time or on weekends, because the smell of 80-pound lots of fresh shark liver boiling in acetic acid -- vinegar -- was so pungent. "People are vomiting in there, Michael," he complained.

But Dr. Zasloff was happy. As I helped with his third prep, he sang something he imagined might be the "Song of the Volga Boatman" while stirring his noxious shark liver soup. "This is one of the greatest achievements of my career," he said, as we wrestled the full garbage cans outside for three days of gravity sedimentation. "I'm doing science on a loading dock with pots and pans and garbage cans. And at the end of it all, I walk in with a little bottle of pure white, crystal squalamine, and people look at it as though I'd ordered it from the Sigma catalog. They don't understand what an incredible triumph it is."

"Teachers make mistakes, books can be wrong, but nature never lies," Dr. Zasloff once told a seventh-grade science class. "If you ask nature the right question, it will always tell you the truth." His notion of great science is a dialogue with nature.

Industry focuses on objectives, while science follows leads. Pharmaceutical research works toward the optimization of molecules, generating and screening huge numbers of analogs of a promising compound in search of the best fit for a predetermined purpose. But the optimization process works only as well as the choice of problems will allow. Like a robot programmed to crawl up a hill and stop at the top, it cannot back down in order to scale other, possibly loftier, peaks. An antimicrobial is not optimized as a cancer drug, nor an antiviral as an appetite suppressant.

Squalamine was a great antibiotic in petri dishes, but it failed to work in animals because of an excessive affinity for serum proteins. The company felt vindicated in its skepticism, but Dr. Zasloff would not let go. Nature, he thought, does not go to the metabolic expense of creating abundant supplies of such a wonderful molecule without having something in mind. And in fact, just as squalamine was fizzling as an antibiotic, a researcher in Massachusetts noted that squalamine resembled certain agents being studied as treatments for cancer.

Years earlier, Judah Folkman at Harvard University had formulated the anti-angiogenesis theory -- that tumors need new blood vessels to survive, and that one could kill them by preventing those vessels from growing. The challenge lay in finding substances that would do this. Dr. Zasloff, with the collaboration of academic colleagues around the country, began testing squalamine and found it extraordinarily potent, with a remarkable mechanism of action.

Cell membranes contain ion pumps that maintain an appropriate internal environment. One family of these, the sodium-proton antiporters, pump out hydrogen ions liberated when the cell grows. If the pump is blocked, a normal cell begins to turn acidic and stops growing. But in tumor cells or virally infected cells [or the abnormal blood vessels underlying wet AMD –Dew], the braking control is broken, and they don't stop growing, but continue to take on acid until they die.

Traditional chemical therapies differentiate good cells from bad by some chemical feature, like recognizing enemy soldiers by the color and cut of their uniforms. The problem is that many microbes can change their clothes. Dr. Zasloff realized that the aminosterol strategy was entirely different: it didn't care what the cell was, as long as it obeyed. When squalamine says stop, you stop or you die, like a game of red light/green light to the death. It cast the differentiation between friend and foe, good cell and bad, in a wholly new light: "If you are under my control, you are me. If I do not control you, I will kill you."

Dr. Zasloff spent long days and weekends working alone in his lab. He found that tadpoles, his old friends from the magainin research, were perfect study subjects. Their half-juvenile, half-adult, nearly transparent bodies allowed him to study the cellular effects of six other, less-abundant shark-liver aminosterols. One made the lining of the gut slough off, one caused muscles to dissolve, one attacked the skin, one disrupted melanocytes (pigmented cells), and two others affected the blood supply to tissues. He saw his tadpoles as a Rosetta stone and spoke of "God's tool kit" -- nature's way of taking animals apart, one organ at a time. He was dizzy with the scientific implications and therapeutic possibilities, though temporarily stumped about how to follow through.

Dr. Zasloff's enthusiasms raised his stock in the lab but not in the office. Whereas other scientists knew how to take it when Dr. Zasloff rushed in claiming he'd just found a cure for cancer, AIDS, or rheumatoid arthritis, his corporate handlers did not. Directors of the company didn't appreciate his dragging them out of a board meeting to peer through a microscope at dissolving tadpoles. While the company was trying to focus on the complex exigencies of completing clinical trials and filing a new drug application, Dr. Zasloff ran wild in a dozen different directions. People were scarcely amused when Dr. Zasloff liberally distributed the batches of buttons he had made up to celebrate his discoveries -- notably a giant button showing squalamine's structure. Even he admits that his self-awarded medals rivaled those of a Russian general. "What's next?" groaned one of his colleagues. "Bumper stickers saying 'Honk if you think I've cured everything'?"

The fate of all these disparate projects -- Magainin Pharmaceuticals' and Dr. Zasloff's, in common and apart -- has turned out rather strangely. Twice, MSI-78, later given the generic name pexiganan and the trade name of Locilex, failed to win FDA approval, not through any fault of the science or its lack of usefulness as a drug, but because of unfortunate mistakes and a lot of bad luck.

A first set of clinical trials used topical Locilex to treat the skin disease impetigo. The trial was precisely modeled after trials done by SmithKline Beecham for Bactroban cream ten years earlier, which seemed the simplest and safest way to go. In the end, though, when the trial was unblinded in April 1994, the placebo performed as well as the drug, showing that proper skin care with soap and water was all that impetigo required. The puzzle was that Bactroban had seemed to succeed, not that Locilex failed.

Dr. Zasloff groaned, "$10 million to prove the value of soap. But at least it's safe. And the science is still good. This is a setback, but that I can stand. If the science was wrong, I couldn't go on."

Five years later, a second set of clinical trials compared topical Locilex to a standard drug -- oral ofloxacin -- for the treatment of infected diabetic foot ulcers, which are responsible for 50,000 amputations in the United States each year. Though the results looked good, the FDA rejected Magainin's application out of concern over manufacturing issues and because an advisory committee had insisted that it wanted a placebo-controlled trial, though it would be ethically difficult if not impossible to prescribe a placebo for such a serious disease. Magainin Pharmaceuticals and SmithKline, its marketing partner, are now trying to determine whether new trials can be devised that will satisfy the FDA's requirements.

Squalamine, that bastard child of garbage-can science, entered Phase 2 human clinical trials for the treatment of solid tumors in 1999; other members of the family of shark aminosterols are also being studied for other applications. One, MSI-1436 [a.k.a. Trodusquemine] originally looked like an antiviral, and Anthony Fauci's lab at the NIH began investigating it as an AIDS treatment until it caused precipitous weight loss in monkeys. Dr. Zasloff, wondering why the animals lost weight but were otherwise perfectly healthy, began studying the mechanism. He has tried the compound on himself, losing 15 pounds in eight weeks, with no observable ill effects. A paper on the compound is due out soon.

In the end, Dr. Zasloff found doing science within a drug company very frustrating. His usefulness to the company on a daily basis seemed limited, at best. Meanwhile, he could not easily pursue projects he thought were genuinely important, if they weren't related to compounds in the company's pipeline.

In September [2000], Dr. Zasloff left the company founded on his discovery, though he remains a board member and scientific consultant. On the evening of his departure, I stopped at his house, and he dragged me up to a small, third-floor bedroom where he had set up a microscope to pursue his current obsession: a substance that primes the body's own natural defenses, taking a very different approach to the control of disease.

"This is really all I need," he said, as we peered at scrapings from his mouth and mine and his wife's and daughter's, some treated with the substance and bacteria free, others not. "Just think, in this day and age -- a scientist publishing papers out of his own attic."
<<