"There's endotoxin in there, you can bet on it," he says, slipping the tube back into a rack.
Hochstein, former deputy director of product quality control (he retired last Sept. 3) in the Food and Drug Administration's Center for Biologics Evaluation and Research, is demonstrating a simple analytical test. It's one that medical professionals, drug companies, pharmacies, and others use worldwide to detect the presence of endotoxins--dangerous toxic byproducts of "gram-negative" bacteria such as Salmonella and E. coli.
The test is the limulus amebocyte lysate assay and is, Hochstein says, "remarkable" for its origin: the horseshoe crab. The limulus test, along with an osteoporosis treatment derived from salmon and a bone filler made from coral, are approved medical products that come from the sea.
Until recently, virtually all medical products had terrestrial sources. For example, organisms found in soil have yielded products such as penicillin, amoxicillin, and other antibiotic compounds responsible for saving millions of Americans from suffering and death.
Sea-based products are rare, but some experts say the world's oceans and waterways may harbor the next generation of drugs, biologics, and even a few medical devices. Dozens of promising products, including a cancer therapy made from algae and a painkiller taken from snails, are in development at research laboratories right now. Other products, such as an anti-inflammatory drug extracted from an organism called the Caribbean sea whip, are under FDA review. Three approved products already have brought the healing power of the sea successfully into the world of public health.
A Lucky Horseshoe
Along the Eastern Seaboard of the United States, it's not unusual when strolling on the shore to find horseshoe crabs that have "beached" or shed their shells. These crabs, the limulus species, are important players in the ecology and marine life of shore areas from Maine to Florida. Their importance increased when, more than two decades ago, researchers discovered that, due to some unique properties, the crabs' blood could be used to detect dangerous endotoxins in drugs, medical devices, and even water.Endotoxins are produced when E. coli and other gram-negative bacteria break down. The effect on humans exposed to the toxins ranges from fever to hemorrhagic stroke. "This underscores the importance of the test in finding these toxins before they can do any damage," says Hochstein.
Before the limulus amebocyte lysate (LAL) test was marketed, medical professionals gauged endotoxin presence by injecting the substance being analyzed into a rabbit's ear. If the animal developed a fever, endotoxins were present. Rabbit tests still are done but are "falling out of favor," says Hochstein, because "they are just too complicated." The tests take four to five hours, and labs must keep caged rabbits on hand.
By contrast, the LAL test uses a glass tube and takes only one hour. Drawing blood from horseshoe crabs causes the animals no harm, and they can be returned to their habitat within 48 hours.
By many accounts, the discovery of the LAL test was serendipitous. In 1971, National Institutes of Health researcher Jack Levin was studying various marine animals when he discovered that blood in horseshoe crabs exposed to E. coli bacteria had clotted. He then drew fresh blood from some horseshoe crabs and exposed it to E. coli in the laboratory. The blood clotted to a gel-like consistency. Further experiments in the NIH Bureau of Biologics, which later became part of FDA, confirmed that if any endotoxins are present, the blood will clot.
Hochstein was a major participant in those early tests, and he recalls setting up shop at a NASA facility on the Eastern Shore of Virginia to catch and draw blood from 1,000 horseshoe crabs at a time. He and his colleagues also kept as many as 200 crabs in tanks filled with ocean water in labs outside Washington, D.C., to ensure an available blood supply.
The team ultimately developed a method for separating amebocytes, which are similar to human white blood cells, from the rest of the crab's blood. These cells then were spun in a centrifuge to intentionally rupture them and create a "lysate," the essence of the LAL test, which is freeze-dried and looks like grains of salt.
In 1973, FDA published regulatory guidelines for producing the LAL test, and in 1977, the agency licensed the first LAL product to Massachusetts-based Associates of Cape Cod. Five other companies have developed their own LAL products since then. Hochstein says FDA's LAL work is an excellent example of transferring technology from the public to the private sectors.
The test has a large market in drug companies that use LAL to detect endotoxin contamination in injectable products, says Melissa Juntunen, marketing coordinator for Associates of Cape Cod. "Probably every major pharmaceutical company uses it," she says. Medical device firms also use the test to ensure that catheters, pacemakers, and other invasive devices are endotoxin-free.
From Fish to Pharmacies
Osteoporosis, a crippling disease marked by a wasting away of bone mass, affects as many as 25 million Americans, 90 percent of them women, at an expense of $10 billion a year, according to the National Osteoporosis Foundation. The disease may be responsible for 1.5 million fractures of the hip, wrist and spine in people over 50, the foundation says, and may cause 50,000 deaths. Given the pervasiveness of osteoporosis and its cost to society, experts say it is crucial to have therapy alternatives if, for example, a patient can't tolerate estrogen, the first-line treatment.Enter the salmon, which, like humans, produces a hormone called calcitonin that helps regulate calcium and decreases bone loss. For osteoporosis patients, taking salmon calcitonin, which is 30 times more potent than that secreted by the human thyroid gland, inhibits the activity of specialized bone cells called osteoclasts that absorb bone tissue. This enables bone to retain more bone mass.
Though the calcitonin in drugs is based chemically on salmon calcitonin, it is now made synthetically in the lab in a form that copies the molecular structure of the fish gland extract. Synthetic calcitonin offers a simpler, more economical way to create large quantities of the product.
FDA approved the first drug based on salmon calcitonin, Calcimar, an injectable form marketed by Rhone-Poulenc Rorer, in 1975. Since then, two drugs made by Novartis and marketed under the trade name Miacalcin--one injectable form and one administered through a nasal spray--were approved. An oral version of salmon calcitonin is in clinical trials now. Salmon calcitonin is approved only for postmenopausal women who cannot tolerate estrogen, or for whom estrogen is not an option.
A Coral Performance
Scuba divers and snorklers have long marveled at the intricate patterns of coral reefs in the Pacific, Caribbean, and other exotic locations. These patterns are now a marvel for people with certain kinds of bone injuries. A product made from the rigid exoskeletons of marine coral can fill voids caused by fractures or other trauma in the upper, flared-out portions of long bones.Called hydroxyapatite (HA), the material is similar in structure to human bone. FDA approved the HA product Pro Osteon Implant 500, made by Interpore International, in 1992. When HA is implanted into a bone void, its web-like structure allows surrounding bone and fibrous tissue to infiltrate the implant and make it biologically part of the body.
The implants, which are either blocks in pre-cut sizes or granules used to fill in the spaces not covered by the blocks, must be used with reinforcement devices such as steel rods to ensure that the fracture remains stable until it heals. "Otherwise," says Nadine Sloan, biomedical engineer in FDA's restorative devices branch, "the implant may crack when you walk or put any weight on it. It wouldn't have sufficient strength to support the weight until bone grows into it or the fracture heals."
Although it is possible for patients to donate bone from other sites on their body to repair a fracture, this causes extra trauma, says Sloan. "One of the real advantages of using [coral-based] implants is that they avoid a second surgery that would be necessary if a donor site is used."
FDA also has approved coral-derived implants for applications such as bone loss around the root of a tooth and in certain areas of the skull.
On the Horizon
Research into new products from the sea, including medical products, is in "high gear" in labs across the United States, says Linda Kupfer, program officer for the National Sea Grant College Program. A unit of the Commerce Department's National Oceanic and Atmospheric Administration, Sea Grant is a network of 29 university-based programs in coastal and Great Lakes areas that involves more than 300 institutions. Though research into medical products is only part of the program's focus, some "very promising work" with medical potential is under way in Sea Grant-supported labs, Kupfer says.For example, researchers at the University of Hawaii have created what may be a novel cancer treatment from blue-green algae. Using compounds called cryptophycins extracted from the algae, researchers have treated mice implanted with cells that cause prostate and breast cancer. The compounds appear to affect the cancer cells' internal structure, possibly keeping the disease from spreading. Much work remains before a drug treatment could be created, but at least one major pharmaceutical company has shown interest in developing the compounds as an anti-cancer therapy.
At the University of Rhode Island, professor Yuzuru Shimizu is developing a culturing system that will ensure an adequate supply of sea-based organisms that show anti-tumor properties. Shimizu is examining metabolites of single-celled plankton called dinoflagellates, which National Cancer Institute tests have shown to have cancer-fighting potential.
Scientists at the University of California's Santa Barbara and San Diego campuses are researching compounds called pseudopterosins. Extracted from the Caribbean sea whip, a type of coral that resembles shrubbery on the sea floor, the compounds are being investigated for use in skin-care products. They also appear to have anti-inflammatory properties and could see use someday as treatment for skin irritations resulting from injury or infection. One pseudopterosin-based product, licensed from the university, is in clinical trials now. The researchers hope to take their work even further: "Our next attempt will be to develop drugs for inflammatory diseases such as arthritis and asthma, among others," says William Fenical, an organic chemist at UC San Diego.
Other important sea-based medical product work is in progress outside the Sea Grant program. For instance, the National Cancer Institute is sponsoring clinical trials of five substances derived from marine invertebrates such as sea hares and bryozoans that may have use in the future as cancer treatments. Elsewhere, one drug company is testing a neurotoxin obtained from a seagoing snail common in the Pacific as a potent painkiller. Early clinical trials have shown that the substance relieves some of the worst kind of chronic pain and could someday be an alternative to morphine.
For the time being, the sea's potential as a medicine cabinet remains largely in the realm of experimentation. But science is moving quickly, and many experts say the world's waterways may soon yield some effective medical treatments, if not some miracle cures.
FDA / FDA Consumer
