INGAP AS A CURE FOR DIABETES

An Update on INGAP and Islet Regeneration

The following is an edited transcript of the special research presentation by Dr. Vinik, Research Director of the Strelitz Diabetes Institutes at EVMS, for the Diabetes Institutes Foundation on March 5, 2002 at Eastern Virginia Medical School.

The overall research objective at the Strelitz Diabetes Institutes towards a cure for diabetes is to find the factor or factors that would allow regeneration of the pancreas.

There are many approaches to curing diabetes: pancreatic transplantation, islet transplantation, engineered beta cells, virtual pancreas, and islet regeneration.

When we began investigating islet cell regeneration at the University of Michigan in 1983, it was generally believed that the pancreatic islet was a terminal cell. However, our research team believed that it was possible to regenerate new islets in the pancreas by reawakening pancreatic adult stem cells.

In Type 1 diabetes, there is an insult on the pancreas that affects the beta cells’ genetic susceptibility, the development of antibodies, and the viral assault. There is a progressive destruction of the pancreas’ ability to make insulin and a reduction of beta cell mass until only about 2% of beta cells remain, and diabetes develops.

In Type 2 diabetes, although a person may start with resistance to the action of insulin, over time the capacity to make insulin is lost. Type 2 diabetes never develops if the pancreas can make enough insulin to compensate for insulin resistance.

From prospective studies in the United Kingdom, we have learned that from the day that a person is diagnosed with Type 2 diabetes, they probably have had the condition for at least 10- 12 years and have already lost 50% of beta cell function. Beta cell loss continues at a rate of 3.5% - 5% culminating in a loss of all beta cell function within ten years from diagnosis.

Because in both Type 1 diabetes and Type 2 diabetes there is defective pancreatic beta cell function, we have proposed that beta cell regeneration could potentially be a treatment for people with people with Type 1 and Type 2 diabetes.

There is a curvilinear relationship between how much insulin a person’s pancreas needs to make depending upon how sensitive the body is. If either the pancreas does not make enough insulin or the body becomes resistant to its action, diabetes then develops. The objective of our research program is to take people who have fallen off that curve and enable them to make more insulin or make them more sensitive to the insulin. We then get them back on the curve again so that they are restored towards normal.

One research avenue to make this possible is islet transplantation. In the past year this process has been highly publicized because of the islet transplantation success of the research group at the University of Alberta in Edmonton, Canada. They have argued that they can improve on islet isolation, they can include larger islet doses immediately after islet isolation, and they can use the appropriate drugs so that the islets are not rejected.

Can this be a truly successful program for millions hoping for a cure? Through this process, scientists will harvest the pancreas from a donor. Then, they will introduce those donor islets into a recipient’s liver, and those islets will begin making insulin normally. This sounds terrific, but we must look at the reality of the situation.

There are about 5,000 pancreases that become available each year. 2,500 of them are already committed. About 2,500 may be available for islet transplantation. It takes 3–4 pancreases to yield sufficient islets to treat one person; it can only be done with advanced disease; and, it requires a lifetime of immunosuppressive treatment with toxic drugs. The immunotherapy may be worse than insulin and is potentially harmful. 2,500 pancreases divided by the number of pancreases needed to yield sufficient islets only allows enough islets to be available each year to treat about 600-700 people. There are 16 million Type 2 diabetics in the United States. There are 1.5 million Type 1 diabetics – many of them children. Do we really want to use this approach with children? We have to consider an alternative approach.

History of INGAP Research

In the 80’s, our scientific team started out on a heretical journey by asking what would be the perfect world to create new islets?

We proposed to regenerate islets from endogenous adult stem cells in one’s own pancreas. These islets could express the full complement of the hormones that were needed – insulin and glucagon – so that in a person’s system, blood sugar would be lowered as well as raised when needed to prevent hypoglycemia. There would be no need for immunotherapy, and we could use benign drugs. This approach could treat both Type 1 and Type 2 diabetes. There would be sufficient insulin production to combat the diabetes as well as the resistance to insulin. Insulin secretion would be regulated. The effect would persist beyond the treatment period. The treatment would not be associated with any toxicity whatsoever. And we would target only the adult pancreatic stem cells. That was a very tall order.

This mythical approach to the treatment of diabetes – recapitulating the normal development of the pancreas in the fetus - has actually now become a reality.

In the ‘80’s our journey began with what we call “sarandipity.” Dr. Lawrence Rosenberg, now at McGill University, and I were conducting experiments to induce pancreatitis. During these tests, we wrapped hamster pancreases with saran. This process led to a completely unexpected discovery – instead of damaging a pancreas, it produced new islet generation. We continued to follow this new line of research and demonstrated that the saran wrapping reversed streptozotocin-induced diabetes in hamsters. At that point, we realized that we had discovered an active principle that we called “ilotropin.” We spent years working on that protein, trying to isolate it to its pure form so that we could administer it to animals made diabetic and later to people with diabetes. The nature of ilotropin eluded us. We had to change the process around.

We had to look within the pancreas that was growing again for a protein that was capable of stimulating new growth. A new technology became available that enabled us “shake the genetic haystack” and watch the needles, or proteins, drop out. We found the genetic message and then went after the gene itself. And in doing that, we discovered INGAP (Islet Neogenesis Associated Protein). Dr. Ronit Rafaeloff showed that the protein product was capable of stimulating islet neogenesis and lower blood glucose levels.

This finding was published in 1997 in the Journal of Clinical Investigation. We found that INGAP has 766 bases. The path that we’ve become interested in is a mature protein. We created the protein by recombinant (molecular biologic) techniques, and then we investigated if this new construct could treat and reverse diabetes in animals.

We gave this recombinant form of INGAP to animals made diabetic with streptozotocin. Several things happened. It stimulated pancreatic duct proliferation; it turned out to be the major component of ilotropin; and, antibodies to INGAP could neutralize the effect. Then we asked if INGAP caused the formation of new islets. Low and behold, it did. We could have been on a wild goose chase. We’d been investigating a product called ilotropin for fifteen years, and we couldn’t find its the active ingredient. Now, after pursuing a gene that’s expressed, it turns out that that gene encodes a protein that is buried in the protein mixture that is doing what we want it to do. We got lucky!

From our work with hamsters made diabetic, we found that for every log dose increase of INGAP, there was a progressive reduction in blood glucose concentration. Each dose we gave dropped the blood glucose 35 mg per deciliter translating into about a 1% drop in blood hemoglobin A1c. It was then just a question of how the process worked, what was the biologic effect, and how often we could repeat the outcome.

By repeating the process, we were able to reverse diabetes 30 – 40% of the time.

Next, we began investigating if a protein fragment could successfully duplicate the action of the whole INGAP protein. We cut up the protein into little pieces to find a smaller protein, a string of 15 amino acids that could do the same work. We synthesized this new INGAP Peptide in vitro and began further testing to see if the INGAP Peptide would reach its target and stimulate islets in the normal hamster.

In an attempt to simplify the problem, Drs. Gary Pittenger and Dr. David Taylor-Fishwick, directors of the SDI laboratories, produced a synthetic peptide fragment of the material and gave it intraperitoneally with a fluorescent tag. They watched where it went in the body. The results were incredible. The injected material went straight to the pancreas and the ducts, and it didn’t go anywhere else. Nature has made us very lucky – it has given us a lock that is present in this pancreatic cell. No matter where you put in the INGAP Peptide, the “key” will find the lock and hone in on it.

Subsequently, we found that once the Peptide got to the pancreas’ ductal cells, it stimulated them to make new islets. We then knew that the biological activity of INGAP was capable of stimulating new islets. We had the answer - INGAP Peptide goes where it is supposed to go and reverses diabetes like the whole INGAP protein.

We moved on to investigate if this Peptide could reverse streptozotocin-induced diabetes in another species. We used the C57BL/bt black mouse that when made diabetic gets inflammation of the pancreas, and the cells look exactly like a person with Type 1 diabetes. We began to investigate if increasing the dose could attain a greater effect. The answer was, yes, in a small study with eight animals. With the animals that received salt water – nothing happened, the diabetes remained. With all the animals that received INGAP Peptide, the diabetes was reversed.

Our next step was to ascertain how the INGAP Peptide reversed diabetes. With the animals that received saline treatment, the blood glucose remained elevated. With those that were treated with INGAP Peptide, the blood sugars started coming down. Stop giving INGAP Peptide, and the blood sugar stayed down. That means it’s not insulin that was producing the effect because insulin only works while it’s around in the body. INGAP Peptide had a biological effect that went beyond the effect of insulin. The Peptide had to have a biological effect to create new cells in the body that make insulin – new cells that the body recognized as its own.

INGAP Peptide In Human Trials

In 2000 GMP Companies became our biotechnology partner with one interest in mind - to make INGAP into a drug to treat diabetes. We worked together conducting trials designed to test dose, safety, efficacy, route of administration, which animals to use, and whether to use humans. We also had to ascertain the Food and Drug Administration’s requirements. It is a complicated, expensive process to take a substance in a test tube and make it into a drug.

Our animal studies showed the drug was well tolerated in mice and dogs. We concluded that it was safe to give animals and likely to be safe to give to humans.

On February 12, 2001, we met with the FDA for a preliminary review of our proposed program. In July we submitted the formal application and received FDA approval in September. On December 5 the human trials began.

Phase 1-2a human trials are being conducted at this time at three locations in the United States. At the University of Texas Diabetes Center in San Antonio, Dr. Ralph DeFronzo, director; at MedStar Institute in Washington DC, Dr. Robert Ratner; and at the University of North Carolina’s Diabetes Center, Dr. John Buse, director.

The Phase 1/2a trial is conducted in two stages. Stage 1 is comprised of administering increasing single doses of INGAP Peptide to 30 insulin deficient patients, both Type 1 and Type 2. Stage 2 is comprised of 34 days of administration of INGAP Peptide. There are 32 patients in Stage 2. The primary objective of these studies is to evaluate the safety and tolerability of single and multiple doses of intramuscular INGAP Peptide administered for the first time in humans.

In these studies we will also be investigating how the INGAP Peptide is disposed of in the body and then evaluating the potential trends toward efficacy. The term “trends toward efficacy” means that if after the first go-around, we’re not successful, our efforts are not ended. If there isn’t a trend, it may be that we need to increase sample size or increase the dosage in a subsequent trial.

New Era for Research - The Strelitz Diabetes Institutes’ Future

There is a whole new era that is ahead of us.

We have come to a fork in the road. This fork represents one branch seeks to develop INGAP as a drug, and the other branch seeks to investigate the future potential for islet regeneration. I say that if we come to a fork in the road, we take it.

We can’t put all of our eggs in one basket. If we say that INGAP is going to be a cure for everyone with diabetes, we may be wrong. So what are the questions that we must be asking, and where do we go from here?

Prospects for the Strelitz Diabetes Institutes’ Research

We want to know what controls INGAP. What are its factors - because we may use these factors to stimulate an individual’s own production of INGAP. What does INGAP control – because somewhere down the line we may find another molecule that INGAP turns on and then we can use that molecule or a smaller molecule or even find the receptor – the key to the lock. We also want to develop transgenic models to show that if a person has an INGAP deficiency, he does have the appropriate islet mass, or if he has an excess of INGAP, he is overproducing it. We will then develop new therapeutic targets. As we develop these new assays (experiments) in the laboratories, we are applying for their patents.

These assays may reveal predictive screens - they may be able to tell us who is deficient in INGAP; who needs INGAP; who is likely to respond to INGAP; who has antibodies to INGAP and who is unlikely to respond to INGAP.

The Role of the Diabetes Institutes Foundation

The Foundation has allowed us to discover INGAP and to explore further - to find that INGAP is present in the human pancreas and to find that it can affect pancreatic tissue in vitro.

We found from our research of patients with chronic pancreatic disease that new ducts are formed and new islets are created when INGAP is present. We were able to conclude that the INGAP that we found in hamsters is the same INGAP that is present in humans.

Our work with INGAP in vitro may have a tremendous impact on the islet transplantation because we may be able to expand in vitro the limited number of islets that are available from donors. We may be able to stimulate donor islets to differentiate and proliferate and prevent the problem of cell death (apoptosis).

Now that idea just recently seemed heretical too. But we have found from our studies that if we add INGAP to islets in tissue culture, the cells start changing to look like intact islets. If we continue to treat with INGAP, the islets becomes more like a pancreatic islets that make insulin. We have been able to do this in the test tube. There is the real possibility that not only will our program be important to treating people, but also will allow us to expand islet numbers.

To develop this avenue of basic science research, Dr. David Taylor-Fishwick is now looking for sites on the INGAP gene that we may be able to stimulate. He’s found and patented two different agents that can help in the development of this process.

We also want to investigate if INGAP stimulates a number of other genes and proteins? Very sophisticated proteomic technology will allow us to look at INGAP’s genetic footprints. These genes that interact with INGAP will be the subjects of future investigations.

INGAP Is Only the Beginning of the Story

Here are the challenges that lie ahead.

We want to know who in the general population has a genetic susceptibility. We need to develop the methods and acquire the technology to do evaluate this.

We want to know who is deficient in INGAP so that we can replace it. We have to take look at Type 1 and Type 2 diabetics, those people who don’t have insulin and those people who don’t have enough insulin.

We have to learn about the antibodies. People with Type 1 diabetes have antibodies that destroy the islets. There may be other elements we need to use with INGAP.

There is an important era of genetic investigation ahead of us, because when we take all of diabetes, we know less than 1/10 of 1% of the genetic predisposition. INGAP deficiency or abnormality may be a very important finding.

Will we need to give INGAP by itself or in combination with other factors? In order to make insulin, a pancreatic beta cell requires multiple different signals that are very carefully coordinated and regulated. Maybe INGAP can stimulate the formation of islets, but maybe it is going to require a lot of refinement down the road.

And then, in Type 2 diabetes, will the new islets we create work effectively? Or, will they function as badly as they did before. If so, we may have to use INGAP in combination with insulin sensitizers.

So where are we now? We think that it is not beyond the realms of reason to anticipate that INGAP alone or in combination with other factors, or small molecules that activate the receptor, or gene manipulation will provide a cure for certain forms of diabetes in humans.

We must prepare for contingencies.

Before there is a cure, there will be a host of complications. We need to reach for the stars and have the support from the Foundation to do it.

Information about the Strelitz Diabetes Institutes’ research in neuropathy and the Executive Medicine Program will be featured in the upcoming issue of the Diabetes Dispatch. More information can be found on the Diabetes Institutes Foundation website at www.dif.org/research/