Oral Insulin Possible: Cholera Shares An Ancient Secret

Fasano reports on successful initial tests of oral delivery of insulin and immunoglobulins in the March 15, 1997 issue of The Journal of Clinical Investigation.

Insulin, taken daily by millions of diabetics, and immunoglobulins used to treat immune system deficiencies, have to be injected because they are macromolecules, too big to be absorbed through the digestive system. A tangled network of cellular proteins called tight junctions normally prevents all but the tiniest molecules from passing through the cells of the intestinal walls into the blood stream.

While studying Vibrio cholerae in hopes of developing a cholera vaccine, Fasano discovered a key that unlocks the tight-junction gate, permitting macromolecules to pass through. That key is a secondary toxin produced by the same micro-organism that causes cholera. Called Zonula occludens toxin (Zot), it is a protein that modulates the tight junctions in the small intestine, making the cells of the intestinal wall more permeable. Zonula occludens is Latin for tight junctions.

In studies in rabbits, Zot caused a 10-fold increase in insulin absorption and a 2 to 6-fold increase in absorption of immunoglobulins in the small intestine. In diabetic rats, Zot administered with oral insulin lowered serum glucose to levels comparable to those obtained with insulin injections. They lived as long on oral insulin as they did when it was injected.

"What we have here is a powerful natural system that theoretically can be harnessed to deliver biologically active macromolecules," Fasano said. "Zot seems to be an analog of a normal physiologic process. It’s not a drug; there’s nothing artificial about it."

As so often happens in scientific research, Fasano stumbled across Zot while seeking something else entirely. Working to develop a cholera vaccine, researchers at the University of Maryland Center for Vaccine Development -- where Fasano directs the gastrointestinal pathophysiology lab -- genetically engineered a cholera toxin lacking the bioactive subunit responsible for the severe diarrhea that makes cholera such a killer. They administered the Vibrio cholerae vaccine candidate lacking active cholera toxin to volunteer research subjects.

They didn’t develop full-blown cholera, but they still had some diarrhea. Something was making the tissues of their intestinal walls more permeable. Something was loosening the tight junctions.

Fasano reports on successful initial tests of oral delivery of insulin and immunoglobulins in the March 15, 1997 issue of The Journal of Clinical Investigation.

Insulin, taken daily by millions of diabetics, and immunoglobulins used to treat immune system deficiencies, have to be injected because they are macromolecules, too big to be absorbed through the digestive system. A tangled network of cellular proteins called tight junctions normally prevents all but the tiniest molecules from passing through the cells of the intestinal walls into the blood stream.

While studying Vibrio cholerae in hopes of developing a cholera vaccine, Fasano discovered a key that unlocks the tight-junction gate, permitting macromolecules to pass through. That key is a secondary toxin produced by the same micro-organism that causes cholera. Called Zonula occludens toxin (Zot), it is a protein that modulates the tight junctions in the small intestine, making the cells of the intestinal wall more permeable. Zonula occludens is Latin for tight junctions.

In studies in rabbits, Zot caused a 10-fold increase in insulin absorption and a 2 to 6-fold increase in absorption of immunoglobulins in the small intestine. In diabetic rats, Zot administered with oral insulin lowered serum glucose to levels comparable to those obtained with insulin injections. They lived as long on oral insulin as they did when it was injected.

"What we have here is a powerful natural system that theoretically can be harnessed to deliver biologically active macromolecules," Fasano said. "Zot seems to be an analog of a normal physiologic process. It’s not a drug; there’s nothing artificial about it."

As so often happens in scientific research, Fasano stumbled across Zot while seeking something else entirely. Working to develop a cholera vaccine, researchers at the University of Maryland Center for Vaccine Development -- where Fasano directs the gastrointestinal pathophysiology lab -- genetically engineered a cholera toxin lacking the bioactive subunit responsible for the severe diarrhea that makes cholera such a killer. They administered the Vibrio cholerae vaccine candidate lacking active cholera toxin to volunteer research subjects.

They didn’t develop full-blown cholera, but they still had some diarrhea. Something was making the tissues of their intestinal walls more permeable. Something was loosening the tight junctions.

Fasano wanted to know what it was.

Cholera was never meant to be a human pathogen, the molecular microbiologist explained. It is a swimmer whose ideal environment is a pond. The human intestine is far too warm for Vibrio cholerae; its oxygen-tension level is too low, and its pH (acid/alkali) balance is all wrong. Trapped inside a human body, Vibrio cholerae is doomed.

But people sometimes swallow the water in which the cholera bacteria swim. So, to survive, Vibrio cholerae had to find a way to escape its intestinal prison. The cholera toxin it produces dramatically alters the fluid and electrolytes balance of the intestinal lining, allowing much more water than normal to pass through, water to flush Vibrio cholerae back out where it belongs. That’s the underlying mechanism of the unrelenting diarrhea that plagues cholera sufferers.

Zot is a second protein produced by Vibrio cholerae to make the cells lining the intestines more permeable if the initial onslaught of cholera toxin fails to free the trapped bacteria.

"Through Zot, cholera taught us that the gate from the intestines to the blood stream can be opened and closed," Fasano said. "Zot is a molecule that is able to communicate directly with the molecules that make up the cell walls, to get them to do things they normally do only under physiological stimuli."

The researcher called it "humbling to realize that we must learn from bacteria, but it is experience, not size, that makes the difference. Vibrio cholerae is a smart pathogen," he said. "It has survived at least 3,000 years, and to survive that long, you have to be very smart. You have to learn to interact with your host, to speak the language of its cells. Vibrio cholerae has a lot to teach us."

Zot research has progressed rapidly since Fasano discovered the protein in 1991. He spent three years purifying the molecule and the past year and a half testing it in the laboratory and in animals. He hopes to collaborate with University of Maryland School of Medicine endocrinologists on Phase I clinical trials in human diabetics soon.