Emmanual C. Opara, PhD, professor at Wake Forest Institute for Regenerative Medicine, discusses "The Bioartifical Pancreas - An Ideal Concept to Manage Diabetes After Pancreatectomy."
EMMANUEL OPARA: Thanks, Clancy. OK. So I'm here to talk about the bioartificial pancreas, which is the current focus of research that perform here. I have no conflicts to disclose. So total pancreatectomy may be required. In a few cases, Doctor Klakow's actually given me a [INAUDIBLE] that the national registry indicates that it may be required in about 2% of individuals who have [INAUDIBLE] the HPB axis. So when you take out the entire pancreas, you're going to be creating what we generally would describe as surgical diabetes, which is a condition of absolute insulin deficience. One of the challenges is that you cannot retrieve islets from the pancreas you remove and then give it back to the patient, because of concerns that you may perpetuate the malignance. So the autologous islet transplantation, which you can perform in some cases like chronic pancreatitis, will become trying to get it in a case like this. So the question, then, is how do you manage a condition like this that actually creates two diseases? You have an exocrine insufficiency, because of the removal of the pancreas. At the same time, you also have an endocrine problem. So I'm here to talk about the endocrine part of it, since the exocrine usually satisfied by the enzyme supplementation, which patience get. Why is it turning itself? I was changing it. So right now, there are two current approaches to treating condition of insulin. Absolute insulin deficiency, which is type one diabetes-- although surgical, you know, diabetes-- type one diabetes induced by surgery, is not the same as the normal type one autoimmune disease. In both conditions you have absolute insulin deficiency and there are two current approaches or two treatment options right now. The standard treatment is insulin. And then, of course, in some cases, if you satisfy the criteria, transplantation may be considered. You get the whole organ, pancreas, or you get islet cell transplantation. And I'll specifically be talking about islet cell transplantation. One thing that we know from the diabetes control and complications trial is the fact that, regardless of how you administer insulin-- whether you use standard treatment or you do intensive treatment-- you can control blood sugar well, but you really would not prevent the development of complications. You can slow them down. And that's in contrast to islet cell replacement therapy, where you can actually halt the progression of any existing complication or completely prevent that. And I've listed a number of complications that have been looked at. When the DCCT study was performed, it wasn't clear why you would achieve very good glycemic control and be unable to prevent complications. You know, the studies that came after that indicated that there may be a role for C-peptide, which is a byproduct, obviously, in processing. I mean, a lot of people in this room probably have thoughts during their training or at some points that C-peptide is a waste product of insulin processing. But studies that I will show subsequently to this slide will indicate that it may have a role. Now, this slide illustrates or gives you the polypeptide structure of a pro-insulin, which is the backbone on which insulin is made and released. Now, you see a beta chain and an alpha chain that are connected by two disulfide bridges. And then they connect the peptide for those two chains. Please, pay particular attention on this amino acid here, Threonine, in human pro-insulin. In the pig insulin, this amino acid is substituted by Alanine. And so that's the only difference between human insulin and pig insulin. The reason now I call this to your attention is that I'm going to make reference to it shortly. Now, when insulin is produced in the pancreas, the pro-insulin peptide is cleaved off at this and at this end to release the alpha and beta chain connected polypeptides that we refer to as insulin. And then the other fragment is referred to as the C-peptide. And C being short for connecting peptide. Now, again, as I said, for a very long time we all were led to understand that C-peptide was just a byproduct of insulin process. Now, this paper was published in 1997 in Science indicating that, indeed, C-peptide may actually have a biological role, where the injection of C-peptide in diabetic animals was found to prevent vascular and neural dysfunction in diabetic rats. OK. And immediately after that, people went to perform clinical trials. And, of course, as always some people found efficacy. Some did not. But the overwhelming evidence is that C-peptide actually plays a very significant role in preventing diabetic complications. In a review that looked at all these studies so far that was published in 2009, came out with a verdict that it does have a role. So islet transplantation is really exciting to a lot of people, because it's much simpler than the more challenging surgical procedure of a whole organ transplantation. Now, this slide here illustrates what is done in the case of autologous islet transplantation. The only difference is that the pancreas is not harvested from the same individual, but obtained from a different individual. And so you isolate islets from the pancreas, and then you load them up in a syringe and inject them into the liver through the portal vein. Now, the concept that I'm talking about involves immunoisolation by encapsulation. That is, you try to shield the islets in some kind of capsule that would enable them to shunt or avoid the immune system of the recipient. Now, this concept actually came up because there are a number of barriers to routine islet transplantation. But by far, the two major barriers are the shortage of human islets. There's not enough. And then, the need to use immunosuppressive drugs to prevent transplant rejection. The second requirement, immunosuppression, would be particularly not be something that you want to do to somebody who has a malignancy, if you need to give them a transplant. So I think the concept of immunoisolation which I'm proposing is actually very highly relevant in that setting. Now this slide just illustrates the principle, which is that-- Whoops. I think I better use my own pointer. --which is that you need to enclose the islets in a barrier matrix that has a semipermeable membrane, illustrated here. What we usually use as the metric of support to immobilized the islet is alginate, which is a complex polysaccharide. Now it doesn't have any permselectivity of its own, so we have to use a polymer of an amino acid to provide that permselectivity. But because poly-L-ornithine or all the polymers of amino acids are very highly charged. You need to cover that up with the negatively charged alginate to make it more biocompatible when you transplant. And what you achieve is permissible transport of these molecules but exclusion on these troublemakers that would want to destroy the islet. I always like to make a distinction between artificial pancreas, which a lot of people hear about, and bioartificial pancreas, which is what I'm talking about. There's a difference between those two. The artificial pancreas is just a mechanical device that you use to administer insulin. And the glucose sensor in the case of the artificial pancreas is a computer that is linked to it. Whereas the bioartificial pancreas is a drug delivery device, a medical device so to speak, which consists of a synthetic, a natural biosynthetic polymer like the alginate that I talked about, and then functional islets. So these cells are viable, and they're live, and they can sense changes in glucose concentration and respond appropriately. And this slide illustrates some of the islets that we have encapsulated in our lab. You can see them right [INAUDIBLE]. So when we started working in this area many, many years ago, we did some initial tests to ensure that the encapsulated cells were responding to changes in glucose concentration. I did some small animal experiments. And the idea of immunoisolation by the encapsulation is that you could get the cells from any source. And so we immediately raised the question of which will be the best source of islets for this kind of a device. And, of course, the pig immediately emerged. I showed you the difference between the pig insulin and human insulin, which is just one amino acid. And of course, when insulin was discovered, Eli Lilly got the patent from the University of Toronto, and had pig farms where they started harvesting insulin, which a lot of people still use. There are a lot of reasons that we consider the pig. Living in North Carolina was not one of them. There are scientific reasons that we consider the pig. We have so many of them that are available and could provide enormous sources for as many as you want. And a few of the issues, particularly the concern about transferring viruses that are endogenous to the pig and do nothing-- don't cause any problem there-- transferring to the human might be a problem. That has been examined and there is no validity to that concern. So the people who funded us when we got into this area, were individuals who have diabetes. And when you have diabetes, you're not looking for a cure in 10 years. You want it yesterday. So they invited me and told me that if I was going to convince them that what I was doing was meaningful, that I needed to do studies in primates. And when I returned to my lab and talked to my surgical colleagues, I said, we're going to buy monkeys. And they said, no, we're going to work on baboon. And after initial hesitation, I agreed. A problem is you might see these guys running around in the zoo, but there are not a lot of diabetic baboons running around. So our initial challenge was develop a mechanism for inducing diabetes in baboon. And then, in fact, the procedure that we adopted pretty much resembles the total pancreatectomy that I started out with in this talk. Here, what we did was perform a subtotal pancreatectomy, leaving only about 10% to 15% of the pancreas to satisfy the exocrine requirement of the animals but taking the bulk of the pancreas away. We still had to destroy the beta cells that secrete insulin in the remnant pancreas. And it took us a few months, several injections, to achieve that. And after we managed the animals on insulin for about a month, we tried transplantation two animals. But I'm only going to show you the data on one of the two animals that had the best performance. As you can see here, the glucose level shown in red was the normal glucose level before the animal was made diabetic using our procedure. And when it became diabetic, the blood sugar went up. And for a month, we managed the diabetes on insulin and brought it down to close to normal. And then, we did a transplant and we drew all exogenous insulin. And you can see how we were able to most months achieve better glycemic control than what we could with the insulin injections. And we did other tests to check glucose tolerance in the transplanted animal. And following transplantation the glucose tolerance came to what it was prior to the induction of diabetes. And that's in contrast with a control animal that we managed on insulin all through. You can see these were the normal blood glucose levels before induction of diabetes. And after we induced diabetes, insulin requirements kept going up, even though we were never able to achieve the same level of glycemic control as in the animal that we transplanted. And when we checked hemoglobin A1C's after about nine months, I believe, you can see that the one with the transplant had hemoglobin A1C that was pretty close to the normal one in native animals which we measured. And that was around 5%, in contrast to the animal managed on insulin. This slide shows that we were able to measure insulin secretory capability in the animals using C-peptide. Because the animals were being treated with insulin, the control animals, we could not use insulin measurement as an index of insulin secretion, because of cross-reactivity between the exogenous insulin and the endogenous one. We had many challenges in these experiments that we're doing in the baboons, which I don't have time to go into. But one of the most frustrating ones was inability to recover the encapsulated islets, which we had transplanted in the peritoneal cavity. At the end of one year, we tried to find these capsules. We couldn't find them. From what I was told much much, much later, we were looking in the wrong place, because baboons can stand up. And under gravity, the capsules go down to the pelvic area, and we were looking in the peritoneal cavity. So at that point, I decided that if what we were talking about was going to be a bioartificial pancreas, there were certain criteria that needed to be met before that would be something that would apply clinically. No use of immunosuppressive drugs, which we achieved because none of the transplanted animals received immunosuppression. We had sustained graft function. As I showed you, one of the animals went on to about 12 months. And then, of course, the technical ease of just putting them in the peritoneal cavity made it very attractive. But there was a problem, because we couldn't retrieve and biopsy the transplant. So I decided to reconsider and re-engineer the capsule in such a way that we could put androgenic factors to induce vascularization, to enhance vascularization and then put them in abdominal fat, the omentum, which would not create a lot of invasiveness in the transplant of the animals. So this slide illustrates the omentum that's been exposed. And then, we have the capsules on them. With the androgenic factors that we included for the delivery, you can see very good vascularization of the transplant. So then, with that we had to try again, this new model of transplantation, starting again from the rat. And I'll show you just a few data here. One of the things we also wanted to check in this rat experiment is to be certain that if you got islets from a different rat strain, and then the recipient rat, diabetic rat, was from a different strain, would get you achieve the same level of glucose control, as if you had rat donors from the same strain as the recipient. Now, the question answered on this slide is the green line shows the glycemic level. And we actually only used a very small mass of islets, because we wanted to see if the addition of the growth factors would enhance the performance of the transplant. So the green is allografts and then, of course, the red is the isograft. If anything, you know, the allografts actually performed better in this transplant than the animals. Now, for both allografts and isografts the body weight maintenance that was achieved was much better than the loss of body weight that occurred in animals that were just managed on insulin and did not receive any transplant. Again, this shows that we were able to use C-peptide as an index of insulin secretion. I keep talking about C-peptide as an index of insulin secretion, because you can distinguish C-peptides secretion across species, which is much more difficult to do with insulin, because the insulin molecule appears to be very close across many species. Using the pig, which I mentioned, you only have one amino acid difference. So this slide shows that we're able to measure secretory activity by the transplants. So this is a very quick overview of what we are doing. But I believe that this would be a very ideal treatment option for the patients who are about to undergo total pancreatectomy because of malignance. Thank you.
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