By David N. Leff

Almost since the discovery of insulin three quarters of a century ago, the hunt has been on for an orally available version of the hormone to replace a lifetime of injections.

Impeding this quest was the fear that swallowed insulin would be chewed up in the stomach or gut before it could reach its action station in the bloodstream.

Type 1, insulin-dependent, diabetes mellitus is an autoimmune disease, in which the body's immune system attacks the insulin-secreting beta cells in its own pancreatic islets of Langerhans, which normally produce insulin. Researchers discovered in 1991 that pure insulin fed to diabetes-prone animals mitigated the severity of their incipient disease.

French clinical diabetologist, Charles Thivolet, has designed an oral vaccine consisting of recombinant human insulin hooked up to a portion of the toxin secreted by the Cholera vibrio microbe. Its purpose is not to replace the needle-injected hormone, but to mobilize the body's mucosal immune defenses against autoimmune cells run amok.

"Cholera toxin is a potent stimulator of the mucosal immune system," Thivolet pointed out. "By using its purified B subunit, you enhance the tolerance mechanism, but not the general immunity."

This B fraction of the toxin has the native function of grappling the pathogen to the inner mucous membrane lining the intestine, where cholera wreaks its deadly damage. Thivolet and his co-workers surmised that, attached to an insulin molecule, the truncated toxin would hustle the hormone through that wall to a rendezvous with the gastrointestinal tracts Peyers patches.

Thirty or 40 Peyers patches strewn along the guts outer wall constitute its gateway to the mucosal immune system. There, the antigenic hormone/toxin peptides would alert protective helper T cells to counter-attack the autoimmune anti-insulin onslaught in the pancreas by creating immune tolerance to insulin.

Thivolet heads a research unit on diabetology at INSERM (the French National Institute on Health and Medical Research) in Lyon, France. He is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated April 29, 1997, titled: "A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes."

A mouse that goes by the name of NOD -- non-obese diabetic -- is the animal model of choice for testing anti-diabetic drugs.

The French team fed a minute amount of their insulin-toxin vaccine to eight-week-old NOD mice, in a single dose. A control cohort of rodents got only the toxin in their chow.

Toxin Hiked Insulin Tolerance

Fifteen weeks after treatment, only two of 15 vaccine-fed animals (28 percent) had developed diabetes, compared with seven of 16 (44 percent) of controls. Other trials, with a larger number of mice, yielded similar results.

Thivolet's group then set out to see whether this prophylactic effect was due to the generation of protective helper T cells -- a special feature of mucosal immunotolerance.

T lymphocytes arise in the spleen. The team extracted aliquots of T cells from the spleens of mice fed the insulin-toxin vaccine, and mixed them with T cells from diabetic animals. They co-injected this mix into the blood of pre-diabetic NOD mice.

Only one in seven (14 percent) of these recipients went on to develop the disease.

Thivolet and his colleagues "are now pushing studies of the mechanism of protection in the pancreas and the gut," he told BioWorld Today. "For the cholera toxin-insulin conjugate," he continued, "it's too early -- it's premature -- to say anything on the mechanism, to say that it is the same mechanism, but more potent, than with oral insulin alone."

He explained: "For oral insulin alone, enhance the TH2-type cells that migrate from the gut to the vicinity of the pancreas, namely, the pancreatic lymph nodes, where they are regulated through interleukin-4 secretions are enhanced. For the insulin-toxin conjugate, the mechanism is probably similar."

He foresees eventual human trials of this "very promising type of action, but not before much progress has been made in the manufacture of this vaccine product. Maybe in a few years this could be done."

Unescorted Oral Insulin Goes On Trial In U.S.

Meanwhile, at 350 locations around the U.S., extensive clinical trials of orally ingested recombinant human insulin alone got under way last September, under National Institutes of Health auspices. The study chairman for this national diabetes prevention trial for Type 1 Diabetes is diabetologist Jay Schuyler, at the University of Miami, Florida.

"Our strategy," Schuyler told Bioworld Today, "is daily feeding of capsules containing pure recombinant human insulin crystals -- potentially in perpetuity -- to individuals at risk of Type 1 diabetes.

"So if Thivolet [whose work Schuyler knows well] can do it with a single oral administration," he continued, "and it really does work like a vaccine, that would be rather dramatic, and a pleasant surprise to me. It is conceivable that the use of cholera toxin may improve bioavailability of oral insulin, and in consequence turn out to be more easy to provide, and more effective, than our approach.

"But whether that would change the rate by which our individuals would have diabetes delayed or prevented," Schuyler added, "would need to be established in a study. So Id be agnostic on the question of whether that would be the case."

He went on to observe that Thivolet actually demonstrates the concept of adoptive transfer. Once an animal is fed the insulin-toxin conjugate, he can extract the immune cells from their spleen, and they afford protection to other animals, without having to feed them insulin.

"What he showed is that part of the mechanism works to create an active form of immune suppression that's unique, toward the autoimmune response in the pancreatic islet cells, where it's working."

Schuyler emphasized the advantage of oral insulin, which taps the mucosal immunity, over injected hormone: "It's suitable to change the balance of the body's immune system, which is really the balance between TH1, the destructive, autoimmune force, and TH2, the protective, tolerogenic force. What were trying to do is force things toward the protective side." *