So it's my pleasure to be able to introduce the speaker for this year's J. Douglas Miller Lecture, Dr. Howard Eisenberg. Dr. Eisenberg holds the Raymond K. Thompson Chair in Neurosurgery at the University of Maryland and is well known for his many years of clinical work and research in traumatic brain injury. So I'd like to thank the program committee for giving me this opportunity. Douglas and I were involved in many of the early head injury studies, and we became close friends, so standing up here has special meaning for me. This is the last talk, as you know, of this session, and what I'll do is I'll show you a novel channel, and I'll tell you why it may be an important target for treatment of traumatic brain injury and stroke, but I'll focus on traumatic brain injury. So as you know, the search to find a drug to improve outcome has a long history. Douglas was in Glasgow when the Coma Scale was being developed. He was one of Jeanette's registrars, so he was there really at the beginning. For most of his career, he worked in Scotland, but he was here twice, at Penn with Lankford, and then later on at MCV with Becker and Young, and I see Harry Young in the audience. These databanks codified severity of injury in the details, and they made clinical trials possible. The first trials tested the then three popular hypotheses of secondary injury, namodipine calcium channel blocker, Turalizate, an inhibitor of lipid peroxidation and therefore free radicals, and then Sofatel, an NMDA inhibitor. That was the gladiolide story. None of these trials, as you know, failed, as did the recent trials, as well as the trials, the non-drug trials, which looked at hypothermia and decompression. It seems curious, at least to me, that the impact factor of the journals publishing these trials improved while the trials continued to fail. Although early on in this trial, which was stopped at 98% accrual because of severe adverse events in the drug arm, this trial made it to the Wall Street Journal. So that's a lot of resources, a lot of effort, and a lot of frustration. I was involved in almost all of these trials and felt that frustration. For me, Melville, the guy who wrote Moby Dick, said it, I think, really well. He said, it's a blind, blank wall that butts all inquiring heads at last. So these are some of the ideas about why these trials may have failed and what may have helped these trials, a better understanding of the condition and a better understanding of trial methodology. There's a lot of talk about new ways of analyzing trials, as you know. Many people feel that what was lacking was Phase II studies that closely tied the target directly to the specifics of secondary injury. Nonetheless, this is not a seller's market for new ideas, and still we have to deal with these patients. Mark Simard, a neurosurgeon in our department, found this channel. It became the focus of his lab. The science has been published in high-impact journals Nature and Lancet. During the next 25 minutes, I'll try to convince you that this channel is not only interesting from a scientific standpoint, but potentially clinically important. These are the main features of the channel. It's a cell surface membrane ion channel. And most important, probably, at least for the standpoint of its therapeutic value, is that it's transcriptionally upregulated following injury. It's not present in uninjured tissue. And in addition, it is upregulated in all of the cells of the neurovascular unit, that is, neurons, astrocytes, and importantly, endothelial cells. The regulatory subunit, SUR1, is a high-affinity sulfonylurea receptor. It is the same receptor seen in the beta cell, which is the target for treatment of adult diabetes with sulfonylureas. Once the channel has been formed, depletion of ATP opens the channel. And depletion of ATP is a feature of severe injury. In the open state, sodium enters the cell, and that's followed by chloride and water to maintain electrical and osmotic neutrality. Then the cells swell, and then rupture, oncotic cell death. The contents of the cell pour out into the extracellular space, and microvessels rupture. Sulfonylurea closes the channel and stops these events. The level of glyburide that closes the channel is much less than that required to affect insulin or glucose. So glyburide here has the potential to be a repurposed drug. It started by looking for a way to harvest untransformed astrocytes as compared to astrocytes in culture. The idea was simple. Gel foam was implanted into a cortical stab wound. A week later, astrocytes were recovered. Patch clamping ultimately showed that the channel was novel. But initially, it seemed as though this was the same channel as was seen in the pancreas, a cell surface ion channel opening dependent on depletion of ATP. One of the several ways to define the channel was to see whether these cells expressed SUR1 or bound glyburide, and this is shown here using SUR1 antibodies. And glyburide fluorescent labeling. And then in this cell, labeled with glyburide, shown to be a glial cell by staining with GFAP. So these are the two channels. By coincidence of nature, as I said, they have the same regulatory subunit, but they have different pore units. And this, it turns out, is a potassium channel. And when ATP is depleted, the potassium leaves the cell, and there's hyperpolarization. In the new channel, same regulatory subunit, but the pore units are different. This is TRPM4, and it's a non-selective cation channel. And when this channel is open, sodium, the most prevalent ion obviously, enters the cell, and the cell becomes depolarized. This shows that isolated cells, when looked at with patch clamping and EM, and then given in suspension, they're given sodium azide. Sodium azide is a carbonic, excuse me, it's a cytochrome oxidase inhibitor, and it reduces ATP. So here you see the cell in its natural state. This is a cell that has the, it's an injured cell, but living without depletion of ATP, so the channel is not open. And the surface of the cell, once ATP is depleted by sodium azide, becomes monotonous. The cell swells, and it becomes stretched out, and then the cell ruptures, and the contents of the cell spill out. The idea that up-regulation of this channel can cause a hemorrhagic contusion was tested in a lot of different animal models, and animal models of spinal cord injury. The model here is the cortical, controlled cortical impact model that was talked about in one of the earlier talks. And you can see that the lesion develops over time. It expands on the surface of the brain, and then as time passes, it extends deeper and goes into the hippocampus. The factors that promote the formation of the channel, SUR1 and the pore unit, are sequential and dependent on whether the stress is, for example, hypoxic, as it would be in ischemic stroke, or mechanical. So the whole development of this sequence, that is, development of the channel, is sequential over time, and that's an opportunity. There's a window provided to then, it's an opportunity to treat patients or to attack this channel, at least with drugs. So using this model and looking for upregulation of SUR1 by labeling, you can see at 3 and 24 hours, there's increased labeling at 24 hours. And then these cells, 3 and 24 hours, are co-labeled with vimentin, a label that indicates that these are in microvessels, and here they're stained or labeled for neurons. So you can see at 24 hours, there's considerable upregulation of SUR1 in these high-powered fields. And then in this immunoblot, you can see that sham, compared to controlled cortical impact injury, there's upregulation or excessive production of the protein, the SUR1 protein. This is in situ hybridization using antisense mRNA, and this is the injured ipsilateral hemisphere, and you can see here, the gene is here, but not well seen in the contralateral, the uninjured hemisphere. This is a control that substitutes SENSE for antisense transcript. And the same thing for the other part of the channel, that is TRPM4 upregulated, this time in mouse brain injury. This is the uninjured control, TBI showing upregulation, and then this is injured brain, but knockout for TRPM4, showing no staining. And here in this high power, you can see the uptake or the upregulation in microvessels. And then this, look, attacks the gene regulation. So this is cortical injury, genes not blocked. In this example, showing less injury, the gene ABCC8, which is the gene for SUR1, is blocked by antisense. Same here for TRMP. You can see that the lesion is much less severe. And then here, as you would expect for the channel that's in the pancreas, there's really no effect. And then this quantifies the lesion size. So this is a gene blocked by antisense here for SUR1, showing the lesion is smaller than in the control. And this reduced swelling here was measured by midline shift. Same thing is the case for the TRPM4 subunit. And then this is the potassium channel showing no effect. So Mark then added another piece of information regarding this whole channel when he showed that the channel SUR1 TRPM4 co-localizes with aquaporin 4. And this is the control. So aquaporin combined with SUR1 or TRMPR4 is yellow. So this is the co-labeling indicating that these are co-localized. And then you can see in the high-powered field, you can see this in a microvessel. The aquaporin in the control only exists in the astrocyte end feed along the blood vessel. And this upregulation of or uncontrolled development of aquaporin 4 is called dysregulation of aquaporin 4, and it only occurs after injury. This slide seems to be complicated, but the concept is really simple. And what's being done here is that aquaporin 4 antibodies are precipitating against the major isoforms of aquaporin 4 precipitate the protein seen here. So they're precipitated, and then they're found in this blot by antibodies to TRMPR4. And the same thing is true for SUR1 as seen here. So aquaporin 4 antibodies precipitate this complex. And in the presence of SUR1, the amount of TRMPR4 is increased fourfold. So the bottom line is that the unit SUR1 TRMPR4 and aquaporin behave as a single unit, and the entire thing can be precipitated by antibodies to any subunit of the channel. So this becomes sort of a jigsaw puzzle. You can see the pieces, the regulatory unit, the pore unit, and aquaporin 4. And the best fit for this is this assemblage. So here's the regulatory unit SUR1, aquaporin 4, and the subunit for the channel pore. And in a side view, you can see this. So the channel is here. And then what happens is that sodium goes down the channel after ATP is depleted. And there's a pool of a high osmolarity here. Water is not well conducted through this channel, but there's easy access for water in aquaporin 4, which is a channel that conducts water in and out of cells. So this is actually how the whole channel works. And I'm going to show you now the treatment effect using glyburide in animal models. So this shows blockage of the channel using now glyburide. This is the vehicle-only and vehicle plus glyburide. So this is placebo. And you can see the extent of the injury, much less in the glyburide-treated animal. And this is extravasated blood. And you can see, over time, this is the vehicle extravasation of blood in the injury itself, and then suppressed by giving glyburide. And this shows the injury again here, vehicle alone, glyburide, the extent of injury, much less in the treated animals. And this is the hippocampus. You can see that this disorganization of the cells in the hippocampus and petechial hemorrhages, and the lesion size is much smaller after treatment with glyburide. Many functional tests were used, including the Morris Water Maze. But this shows vertical expiration or rearing. And you can see, over time, after glyburide, the recovery is much greater, while there's no recovery in the untreated animals. Lastly, I'm going to show you two Phase II clinical trials. This trial, Intrust, was sponsored or funded by the Department of Defense through the Army. Remedy Pharmaceuticals did the Phase I dose escalation trial and also provided the drug for this study. So this is a randomized, double-blind-controlled Phase II trial with surrogate outcome measures. And the outcome measures were MRI measures of edema and blood. The subjects were severe to moderately injured patients, but the patients were stratified, according to Glasgow Coma Scale, by center. The window, which was assumed from the animal studies, was 10 hours. So 10 hours from the impact to giving drug. And within that 10-hour period, the patient had to have a baseline MR. So this became a complicated study. The indices for edema were mean diffusivity in tissue and then free water. And these were measured from baseline to the end of the infusion. So the whole study is a 72-hour study, essentially. Blood was measured just by the voxels that show change by SWI. And then the contusion volume was measured in CT scans. There were three regions of interest. Contusions, where we took contusions that were at least 5 cc's and combined them, white matter that appeared to be uninvolved, and then whole brain. And then safety was measured by serious adverse events. The centers were Maryland, Pittsburgh, and San Diego. The Harvard Medical School did the MRI analyses. So about 1,500 patients were screened. Most of the screened values were based on Glasgow Coma Scale and age, which was built into the protocol, obviously, but also perceived in an inadequate time to get the patient stabilized and then into an MR scan and then given drug all in 10 hours. At the same time, we had to find somebody who would give consent. So 1,500 patients, roughly, became less than 40 patients. And these never made it to randomization because the time ran out to give the drug. So we ended up with 29 patients. One patient, the consent was withdrawn before any drug was given. So a small study, but all those patients had drug infused for 72 hours. The splits were luckily favorable with regard to gender, median age, and initial ICP not terribly different. The Glasgow Coma Scale, the most common Glasgow Coma score was 7. So these patients were not the worst patients. And the reason for that probably was because of the difficulty getting the patient into a scanner and then given drug within that short time period. And then we were particularly interested in that region of interest, contusions, because of what was known from the animal studies. And you can also see in the same thing, and I'll show you that in a moment, same thing in the human subjects, that contusion was, these markers were most robust in the area of contusion and the drug effect was greatest there. So for edema, there were nine comparisons. Remember there were three regions of interest and three MR indices, mean diffusivity in tissue and free water. In all instances, things got worse over 72 hours, as you would imagine. So the 72-hour values were worse than the baseline, no surprise. So of nine comparisons, though, in the drug arm, there was more stability. In other words, the slope of that change over the two points, if you can call that a slope, was less. And this was more of a flat line. And in the placebo, things got worse more profoundly. That's in seven of the nine. In one case, there was no, in one patient, there was no difference. So we inferred from that that the drug was associated with stability with regard to edema. The largest change was in the untreated patients with contusions. So that was, with regard to these measures, that was the most obvious injury. Blood baseline to 72 hours decreased in the placebo arm and the drug arm, but much more in the drug arm. These are the data. So this is free water. So free water, more free water compared to baseline in the placebo, less in the drug. Less so in the tissue diffusivity. Mean diffusivity was just really a combination of these reflecting sort of a midpoint. The volume of the lesion changed. This is a negative change. So the change was less here and greater here. So that was a beneficial effect, if you will. And then the same with blood. There was a greater change, a reduction of blood in the contusion in the treated patients as compared to the placebo. And these are the data seen in these plots. So there were no deaths in the study in either arm. There was neurologic worsening, which was a change in motor score of two or a pupil change, and that occurred in two patients in the drug arm and only one in the placebo arm. Then we were very, of course, concerned about the possibility of hypoglycemia. There were two transient episodes of hypoglycemia reaching 50 or less than 52 milligrams per deciliter. There was a very highly developed protocol for dealing with hypoglycemia. And using this protocol, the drug was never suspended or discontinued. The patients were treated. The patients that had hypoglycemia, which were few, were just treated with glucose. And this shows the severe adverse events. And there was one patient greater here in the gliburide arm than in the placebo arm. And in that patient, the excess is here. This is probably due to the drug itself. But remember, this is a small study. So quickly, I'll show you another study. This time for ischemic stroke, more robust, 86 patients. This is the split. These were large strokes. These are strokes that have a high chance of dying. And you can see they're pretty close in average and mean volume. The ages are pretty close. This is the percentage of patients that were given TPA, and that worked out well. And the window was the same as the window for the traumatic brain injury. Thirty-day mortality was less in the treated arm than in the drug arm. So 16% died here, while 30% died in the placebo. And with regard to midline shift, it was more robust in favor of the drug. The midline shift, on average, was slightly more than 4 millimeters. And then the treated, slightly less than 9 millimeters. So here you could see, according to whether this was by protocol or by intent to treat, you can see the differences in mortality. And these are the survival curves. This is the drug, and this is the placebo. So greater risk of dying in the placebo arm. Again, a small study. So this is going on now to a more full-phase study, and that's just the beginning now. So why focus on this channel? One of the main reasons is that it's not present in normal tissue. It only appears after injury. So all of these channels are bad guys. As compared to NMDA, which is present in normal tissue and actually an important part of functioning of normal tissue, and only occurs in neurons. This channel only arises after injury, and it occurs in all cells in the neurovascular unit. So it's much more pervasive. The mechanism has been well-studied. There's a large amount of information of the drug. For 25 years, it was used to treat diabetes. So there's a lot known about the pharmacokinetics. We did pharmacokinetics in the trial as well. So this is a repurposed drug, if it ever becomes a drug for head injury. There's a lot of data from preclinical. There's a lot of preclinical data, not only animal models, but information from cell and sub-cell experiments. And there's two studies so far that show positive trends, albeit they're small studies. So this is the important members, the important players, Mark Simard's lab. This traumatic brain injury clinical trial, David Okonkwo from Pittsburgh was involved. And these are the people who ran the MRI analysis lab at Harvard. And then the stroke study originated in Maryland, but the PI was at Yale, Kevin Sheff. So that team, there were 18 sites in that study. So I think I finished on time. It's 5.30, and we're in New Orleans. And I'm sure people can think of better things to do than listen to me. So thanks for coming, and thanks for your attention. All right, do we have a question? Which of the components of complex, like the right block? It's the regulatory subunit. So SUR1 is a high affinity sulfonylurea receptor. And that's where the… Sodium permeability and water permeability. It blocks… Well, it closes the channel, so it blocks… Well, sodium being the most prevalent cation, it… So if I didn't potentiate, I could not even save the experiment. Say that again, I'm sorry. If I didn't potentiate, I could not even save the experiment. Why? Why? I'm not sure. I'm trying to… The question is why… Might it? Yes. Well, might it? Would it? Would it? Might it? Yes. I'm sorry. Would it potentiate? Yeah, I have no information on that. Would it… Because you're less likely to let the sodium get into… Yeah, I see your point, right. Yeah, I don't know the answer to that. I'm sure some of these… You know, it's a very small study. That's the problem. Probably many of them did get hypertonic saline. So I don't… You know, to look at that and see what the balance was would be tricky. It may not be conclusive at all. This is… Remember, there's only 28 patients in the study, all told. So after all of that, the answer is I don't know. Okay. So we don't know how many of those patients are being managed with hypertonic saline? I could find out. No, I don't know. Yeah. Probably a lot, right? Yeah. So the third question I have… And the same would be true of the big influx, right? It might be easier to look at that than to look at bigger numbers anyway. And the third question is, should we be considering possibly using glycoride for glucose control in the early period? There is a study, a meta-analysis of stroke for centers. And this was a large number of patients. And what they looked at was patients with diabetes that had large influx. And they were all getting oral diabetic, but not all given glyburide. So the split was stroke, diabetes, or oral anti-diabetic drugs, and then split out for sulfonylurea, glyburide, versus other oral. And it was favorable. There was a real difference. I mean, there's only a limited amount of time, 25 minutes to get through all of this. So I didn't show that, but those data are impressive. So, Dr. Eisenberger, I have to ask you, I usually don't think of diabetes as being a good prognosticator after a head injury. You sort of raised the question, you know, about glyburide. Do you think the issue that why we haven't identified it sooner is maybe because we're switching these patients to insulin as soon as they hit the ICU? I don't think. I think until Mark found this channel, nobody heard of this. Because it's not the same channel. You know, the channel in the beta cell is a potassium selective channel. So they're only similar by the fact that they have the same regulatory unit. So I don't think. You know, there's a lot of questions you can ask, like should people who would be exposed to trauma get glyburide, you know, prophylactically? Because it's a very low level. And I don't think you can convince the NFL to give players this drug. But, you know, and we have used it, some of us. We have chief residents here, so they can correct me. It has been used for some patients, but it's not FDA approved for this purpose. So we'll know more after this larger study of stroke. The patent now, so the infusible formulation of glyburide is now owned by Biogen. And they're looking for a head injury study. But people in this room know how difficult that will be. But maybe, you know, stroke I think is a lot easier to study than head injury. We'll see how that turns out. But that's just started, so that information is at least three years away. So thanks again. Thank you.

traumatic brain injury

drug

cell surface membrane ion channel

glyburide

clinical trials

improving patient outcomes

repurposing