Hello, and welcome to today's webcast, Models of Translational Research in Critical Care. Today's webcast is brought to you by Discovery, the critical care research network at the Society of Critical Care Medicine, in collaboration with the CPP section. My name is Matt Wannett, clinical associate professor at the University of Houston College of Pharmacy in Houston, Texas. I have no disclosures. So once again, thanks for joining us. A few housekeeping items before we go ahead and get started, there will be a question and answer session at the end of the presentation today. To submit questions throughout the presentation, type your question in the question box located on the control panel. This webcast is being recorded. The recording will be available to view on the My SCCM website within five business days. There is no CME associated with this educational program. However, there will be an evaluation sent at the conclusion of this program. The link to that evaluation is also listed in the chat box for your convenience. You only need to complete it once at the end of the webcast. Your opinions and feedback are important to us as we plan and develop future educational offerings. Please be sure to take five to 10 minutes to complete this evaluation. And now I'd like to introduce you to our speaker for today. Dr. Sam Poliak is a professor of pharmaceutical sciences and the associate dean for graduate and postdoctoral programs at the University of Pittsburgh School of Pharmacy. Dr. Poliak also serves as the director of the Center for Clinical Pharmaceutical Sciences and the director of the Small Molecule Biomarker Core Lab at the University of Pittsburgh. His work is focused on determining the role of drug metabolizing cytochrome P450 enzymes on fatty acid bioactivation and drug disposition in the pathogenesis of disease and critically ill patients. Research in his laboratory utilizes relevant models of disease and translate these findings to critically ill patients. The NIH and the American Heart Association have supported his research leading to over 74 peer reviewed publications. And now I'll turn things over to Dr. Poliak. Thank you, Matt. I'd like to thank you as well as Caitlin Alexander for the opportunity to present today and everyone from SCCM for making today's events possible for the presentation. I'm really looking forward to presenting the following objectives. The first objective is really to describe the national definitions of translational research and differentiate translational research and the spectrum of translational research from clinical as well as basic science. In addition, I'm going to use examples from my own research and my collaborators' research at the University of Pittsburgh to delineate the importance of pharmacy and pharmaceutical science expertise in clinical and translational research initiatives and provide an example through our work of the ways in which we collaborate to bring our areas of expertise in critical care, our expertise in pharmacy, expertise in nursing together as a collaborative research group. I'll also talk a bit about along the way ways in which we developed our interdisciplinary collaborations across the research that we've conducted. But first starting off with the definition, there's often confusion as to what exactly is translational, what is clinical translational, what is basic science versus preclinical. I think the best definition really comes from the National Center for Advancing Translational Science. This is directly from the NCATS website looking at the definition of the translational research spectrum. Translational research is really not a linear process, it's a spectrum of research that goes back and forth with each one of the lines in this diagram is really being bi-directional. So you can see on the far right, we have the basic science research, which feeds into preclinical, a clinical research, clinical implementation in public health. We usually think of this as a linear process, but the reality is that there's lots of interactive thought that should be going into ways in which basic science research is conducted, keeping in mind what the clinical implementation would be and what the public health ramifications would be of a discovery. And being able to both develop new approaches, demonstrate their usefulness and disseminate those findings to advance clinical and translational research. So whenever we discuss clinical research, I'll provide a definition in a bit. That's one area or one silo, if you will. Clinical implementation is another silo, public health being a silo. Translational is breaking out of those silos and actually having bi-directional thought of how to develop the basic science research the whole way through the public health implications of what new discoveries would yield. So the definition of the translational research spectrum is that each stage of research along the path from the biological basis of health to disease interventions that improve the health of individuals. The spectrum is not a linear process, but actually a bi-directional process where each stage builds upon the other and it forms the other stages. And the patient is at the center of that process, thinking of how this would actually impact patients long-term. I like this definition as well from NCATS, it's the process of turning observations in the laboratory, clinic, and community into interventions that improve health of individuals within the public, which is a simple definition of the translation, but it is everything from the basic science through the public health implications of that work. Breaking into the different definitions of preclinical and clinical research, as well as clinical implementation research, preclinical research are really models to understand the basis of disease and treatment. It's our cell or animal models of disease, also our human and tissue sample evaluation, although the human tissue does tend to translate into what the definition of clinical translational research is, and computer-aided simulations, which is an emerging area of really looking at both drug development as well as disease implications of certain complex pathways of modeling and simulation. Clinical research is when we begin testing those technologies in people and understanding the pathogenesis in humans, and then testing the interventions for the phase one safety and the phase two and phase three effectiveness of those interventions with and without a disease, behavioral observations and studies, as well as the outcomes and health services-based research. Then the clinical implementation is after the clinical research has been demonstrated and the safety and the effectiveness of an intervention is known, actually how that research or how that intervention becomes part of routine clinical care for the population. Implementation of that research and seeing how that implementation is conducted, and then evaluating it and basically figuring out where the gaps are in our care in order to re-inform what the next intervention should be in the preclinical stage to move through the clinical implementation. But each one of these definitions, and I could have added health outcomes and policy as well to these definitions, but the research that I'm going to talk about today are going to be examples really of preclinical research, moving into clinical research and moving back into preclinical research. And all of the work that I present in my own work will take into account what the clinical implementations are. So whenever I get into the actual presentation of the research that we've conducted, it really does move between that preclinical, moving back into that clinical. And why is that important to think of the clinical implementations of the research in these early phases? It's because we have lots of examples of how the spectrum needs to be improved. About 80% of our indications fail in human clinical trials because they're found to be either unsafe or ineffective. There are many reasons for the failures, and there are many problems with drug development. But one of the reasons that has been identified in virtually every publication on this topic is the demonstration or evaluation of adequate drug exposure and target engagement in humans. And this will be a theme that we'll talk about today because this does fall in the expertise of pharmacy, pharmaceutical sciences, and pharmaceutical science expertise and research. So being able to make our therapeutic interventions think not only of adequate concentrations, but adequate concentrations in preclinical studies, how they translate to being able to get adequate concentrations in humans at the site of action, but even more importantly, knowing that whenever you have adequate concentrations of a drug, you're able to see that that target that you intended for the drug to work on has been engaged and actually is effective as being a potential therapy. So in my own research, we really developed a collaborative team. And I'm showing the end result of a collaborative team of researchers, and there could have been many others on this slide. But this is over many, many years, and each collaboration involved a different research project that kind of evolved. It really began with Pat Kohanek as the director for the Saffir Center for Association Research inviting me to a journal club, and I was a faculty member in pharmacy. And I joined a journal club where there were faculty from nursing, faculty from pediatrics, faculty from emergency medicine, as well as critical care medicine and anesthesiology. We all came together around the table, around the topic we all had in common, which is our interest in critical care illness and resuscitation research. Building the team wasn't identifying everybody, creating lists, picking up the phone, and asking them if they want to be on the team, it was building the relationships. And building the relationships one by one and letting them evolve over the time, over the course of the career. And I'll show a bit of our research that we did over the years that it evolved in this way as we move through the talk. So what was the interest area? Well, my personal research interest area came out of my graduate training, and it was really about secondary hyperperfusion and neuronal injury after the initial insult. So within stroke, the difference between the diffusion and perfusion area and collateral blood flow trying to basically limit the diffusion and improve the perfusion in order to decrease the overall brain damage as being a potential target for thromboembolic stroke. In hemorrhagic stroke, the vasospasm and loss of autoregulation and vascular dysregulation is also a common complication, which leads to more complicated or poorer outcomes in those hemorrhagic stroke patients. And my collaborator, actually Mio Manoli, demonstrated in secondary reductions in a pediatric preclinical model after cardiac arrest, even after resuscitation. There were times after V-fib cardiac arrest where there were these secondary reductions in blood flow that were associated with poor neuronal recovery. So I became interested in what the mechanisms are behind secondary hyperperfusion and secondary neuronal injury, and was there a drug target that we could intervene or develop and see if it could actually be developed for therapeutic intervention. My interest also really stemmed from that dysregulation of blood flow. And one of the evaluations by Ling Zhu Li in my laboratory was looking at the no-reflow phenomenon. So this is actually a two-photon microscopy image that was taken in a pediatric rat cardiac arrest model. And on the left-hand side, you can see the capillaries with red blood cells flowing through those capillaries. And this is in vivo after resuscitation for cardiac arrest. The left is at baseline, and the right is 30 minutes after cardiac arrest. And what we observed was a percentage of the capillaries were basically having the no-reflow phenomenon, which had been reported many times earlier, but that's one of the first observations in vivo of looking at and seeing no-reflow in the animal. This no-reflow is actually one of the possible mechanisms, and there are many. But the mechanism we were interested in studying more thoroughly was the conversion of arachidonic acid by cytochrome P450 enzymes to a metabolite known as 20-HEAT, 20-hydroxyacosate tetraenoic acid. The reason I became interested is in my background in pharmacy, I had learned over the years many details, and my thesis research was actually on drug metabolizing enzymes. And cytochrome P450 is better known for the fact that it metabolizes many of our drugs and actually is a pathway of elimination and drug-drug interaction. But I became interested in what the function of cytochrome P450 is in the brain. And one of the things that cytochrome P450 does in the brain is convert arachidonic acid to this 20-hydroxylated metabolite that is known to be both vasoconstrictive, and I should add to this slide, also leads to neurodegeneration and actually decreases neuronal survival in culture as well. So it's both detrimental in terms of neuronal survival and also vasodasoconstriction. So we became interested in studying, in our preclinical phase, what was the role in post-cardiac arrest brain injury, and is this pathway involved, and could we inhibit this pathway to evaluate the role in brain damage after cardiac arrest? So a couple of more details about the pathway that we were interested in studying. We were interested in looking at vasoconstriction, 20-heat mediates, and also neuroprotection. One of the ways in which this is thought to work from a really seminal paper by Hall and Nature in 2014 was evaluating the vasoconstrictive response in the nitric oxide decreases, which is known to happen particularly in dysregulated autoregulation after a subarachnoid hemorrhage. And these reactions in nitric oxide actually increase 20-heat formation rate, and it's this balance between vasodilation of PGE2 and vasoconstriction by 20-heat that mediate cerebral blood flow by changing cerebrovascular resistance. So we became interested in the fact that if arachidonic acid is converted to this neuronal injury slash vasoconstrictive metabolite by CYP4A11 and 4F2 in humans, interestingly, arachidonic acid is also converted by different CYP enzymes to a vasodilatory metabolite, which are the EATs. And I show 11, 12 EAT in this picture, however, any of the epoxides are actually vasodilatory and thought to be neuroprotective. So we have two different drug metabolizing enzymes, but CYP enzymes, these are drug metabolizing, and arachidonic acid or fatty acid metabolizing enzymes that take the same substrate to yield two different phenotypes, neuroprotection versus neuronal injury. So the question became, could we inhibit one of these pathways and see if we can mediate the neuroprotection? And we were fortunate in that there was a non-drug-like compound available for studying this in preclinical studies. So we asked the preclinical research question. So going back to that translational spectrum that I opened the talk with, in our preclinical research, we asked the fundamental question of, does inhibition of 20 heat formation protect the injured brain? And we looked in our early studies, we really evaluated two different models, but in order to evaluate those models, we really needed to reach out and work with our collaborators. So our collaborators' expertise in these cases, both Pat Kohanek and Mio Manoli in the Saffir Center for Resuscitation Research and Pediatric Critical Care Medicine and Anesthesiology, they had expertise in preclinical models of injury. And their true expertise is being sure those preclinical models of injury mimic as close as possible the clinical injury that is observed in patients. They have expertise in traumatic brain injury, which I won't show any work with that today, but also in a cardiac arrest model. And from discussing in Journal Club, we're interested in evaluating in stroke and made the recommendation from Pat Kohanek to go talk to Steve Graham and collaborate with Steve Graham at the University of Pittsburgh and the VA Medical Center and University of Pittsburgh Center for Neurosciences and work with them in their preclinical model of middle cerebral artery occlusion. What we observed in the early studies, first, we needed to know is, does our inhibitor HET-16, which inhibits 20 enzymes, CYP4A2 and 4A11 and 4F2, does the HET-16 inhibit 20 heat formation or does it affect EATS? Because the EATS, again, are protective and 20 heat is detrimental. So we evaluated in vitro and were able to observe that our concentrations of our inhibitor did not affect any of our EAT concentrations, but it did affect our 20 heat. 20 heat was significantly reduced to two different concentrations that we evaluated. The reason I'm bringing this out is this was early work of evaluating the pharmacology in vitro and then in vivo that we conducted to be sure that we had the right drug getting at the right concentration or the right compound, in this case, it's not a drug, but the right compound at the right concentration at the right location in order to inhibit the formation of the target that we aim to inhibit. In this case, the target was inhibition of 20 heat formation and not affecting ancillary pathways, in this case, the EAT formation to be beneficial for our model evaluation. Then we were able to evaluate the injury. This is an early study in our research where we looked at TTC staining. So essentially, the dye turns red if mitochondria are functional. It does not if the mitochondria are not functional. This is a crude assessment of evaluation of neuroprotection, but it was effective at the screening phase. So this was purely preclinical in evaluating if you inhibit this pathway, is it actually a target or not for neuroprotection? And we observed in these early studies that we were able to significantly reduce the overall lesion volume in the rat middle cerebral artery occlusion. And later, we were able to evaluate in our collaboration with Mio Manoli in the prenatal day 17 or the pediatric asphyxial cardiac arrest model. These are the number of pycnotic neurons that we observed on staining. When animals were either treated with vehicle or the HET-16 inhibitor, we were able to observe neuroprotection in these studies, and these were all blinded animal studies and randomized for the evaluation of the neuroprotective pathway. So we had preclinical evidence that the pathway was important, and there were others that did many studies and other models, which I'm not going to share today, but both collaborators and individuals at other institutions that showed that if you were able to inhibit this pathway, it was protective in various different injury models. That led us to the question of clinical research in R20 heat concentrations associated with outcomes in subarachnoid hemorrhage patients. I'm presenting this as if we did all of the preclinical research, and then we moved into the clinical research. The reality is that we were beginning both simultaneously. We were doing the preclinical research in the animal model, and then through collaborations that started from that same journal club with the School of Nursing, we developed our collaborative team to evaluate in subarachnoid hemorrhage patients. So back in the initial start of this work, Mary Kerr was at the University of Pittsburgh. She became the dean at Case Western School of Nursing and is retired now. But Mary and I were attending journal club back when we could attend journal club. We're now attending virtually. But when we were attending journal club in person, we would walk back up the hill, and the nursing school was halfway to the School of Pharmacy. And I presented an article on R20 heat in the journal club. And Mary said to me on the way up the hill, can you measure this in patients? And at that point in time, no one had. But one thing in the pharmacy expertise world that we could do is measure the concentrations of these metabolites. And we had a whole assay panel for assessing that. So she had already had research looking at biomarkers in subarachnoid hemorrhage patients and had a CSF bank of samples they had collected over a number of years. This led to writing my first grant with Mary as the PI, but learning to write grants from her early in the career. We extended that research with Beth Crago and Paula Sherwood, and we were able to lead this research. And it lasted, I believe, a total number of 15 years in evaluating the clinical subarachnoid hemorrhage research and these concentration of these metabolites. Again, it was taking the expertise that we had in pharmacy and pharmacology, the expertise in being able to assess the concentrations and assess the metabolites, and collaborating with individuals that brought their specific expertise on going patient recruitment, biomarker evaluation, and outcomes assessment, and building that collaborative team to evaluate that. So we ultimately conducted a large study. It was a prospective longitudinal design. It was a large cohort of subarachnoid hemorrhage patients. The end study had 353 patients over the entire time frame of the study. The inclusion criteria were subarachnoid hemorrhage diagnosed within five days with a significantly large bleed of Fischer grade of greater than two, and our setting was the recruitment of data was collected in neurovascular ICU at University of Pittsburgh Medical Center Presby Hospital. We evaluated and collected blood and genetic data, as well as CSF biomarker data during the first 14 days or as long as the individual was in the hospital, and also had the diagnostic monitoring for impaired cerebral blood flow during that early period. And then we had long-term follow-up data at three and 12 months in these studies. So we were able to use the expert mass spec instrumentation in my laboratory, so HPLC-MS, and my laboratory to measure and set up the assays for evaluation of all the metabolites, which we already established. And we were able to see that we can indeed measure 20-heat and CSF concentrations. When we finally unblinded the data and looked at the patients, we initially looked at what were the concentrations of the metabolites versus time after injury, and we looked at 20-heat relative to the most abundant heat metabolite. We looked at it relative to all of them as well, because the di-heats are the breakdown products of the neuroprotective, and 20-heat is thought to be the detrimental product. And what we observed was very interesting, because we had a lot of patients that were low, but of the patients that were high, they were all high early after injury. And typically in subarachnoid hemorrhage, that vasospastic event is three to seven days in the peak, and can be even out to later time periods for vasospasm. So we did have a bit of a temporal relationship between increased 20-heat and when we would expect elevations after subarachnoid hemorrhage that may be mediated by that metabolite. In the clinical studies, we really could only evaluate the associations, but in the preclinical studies, we had the clear inhibition of the pathway was involved in other models of injury. And just to close the loop a bit, Richard Roman's laboratory and others have evaluated the animal subarachnoid hemorrhage model, and were able to show neuroprotection in that model as well in the preclinical work. What we observed in this graph, you can see that the maximum 20-heat concentration is reported on the y-axis. This is clinical neurological deterioration where we saw significant relationship. We did not see a significant relationship with delayed cerebral ischemia, but did see a significant relationship and a trend towards significant relationship for MRS at three and 12 months. Whenever we broke out mortality, we observed basically the patients that were in the low trajectory group versus in the moderate or high trajectory group, meaning if you had moderate to high concentrations over time of 20-heat in your CSF, you were threefold increased likelihood of having a worse mortality or mortality at three months. And that was about threefold increase in mortality. That was significantly different amongst the groups. And we observed the 3.3-fold increased risk in the moderate high to high group in the work that we evaluated in subarachnoid hemorrhage. So this research was that movement in some ways, because we were looking at the preclinical evidence that told us the pathway was important, and now we had clinical data that implied that high concentrations were associated with poor outcome, therefore making that link, the pathway, may be important as well as a clinical target. But this led us back to where we are now, which is the next question. What we had was a tool that worked great in preclinical animal models, but was not really a drug. It had a very short in vivo half-life. It didn't have great selectivity. And so we wanted to have a compound that had drug-like characteristics. So we began to look at the inhibitors of the CYP4A11 and CYP4F2, and asked the question is, could we develop this as a target for intervention? Could we develop compounds with drug-like characteristics that could be developed to intervene in this pathway as a neuroprotective agent? This led to the next group of collaborators that we began this work. I believe it was prior to 2015. It was right around 2015 that we began this collaboration with David Coase and Lee McDermott. Lee is in the Department of Chemistry, and Lee is in the School of Pharmacy, but they have medicinal chemistry expertise. David Coase's expertise is in computational modeling, and Lee McDermott's expertise is in chemical synthesis and industry drug development. So he actually served a large portion of his career in the pharmaceutical industry, developing drug-like compounds for CNS indications before joining the academic ranks within our school. So we really had a collaboration that developed over the past six years, now five to six years. And it came, again, out of, in this case, it wasn't a journal club, but it was a presentation presenting our research that we've conducted on the 20He pathway. And Lee literally came up and said, I'd like to develop compounds for you. And we began a conversation after that session, and we started evaluating his expertise in the area. And it turns out he had an incredible wealth of knowledge on how to develop compounds that would cross the blood-brain barrier. And together, we developed a collaborative grant to evaluate these pathways, which is the research we're conducting today. And I'll update you with how far we've taken this research to date in the coming slides. So what we were looking to cross is this pathway of, it's coined the valley of death, a bit of an overstatement, but it's the death of drug development in this case. And it really is the fact that we come up with a lot of drug targets within a silo of preclinical research, or even in clinical tissue evaluation. But the evaluation of validating the target, coming up with lead compounds, developing it preclinically to show that it truly is effective with an intervention with a drug-like compound, to move drugs into phase one or into phase two is where many, many compounds do not get developed. So we have lots of bridges in Pittsburgh. My student had the bright idea of bridging our valley here to actually look at all of the compounds that we were evaluating. And we developed a strategy. And our strategy was to collaborate with both our computational biology folks, our clinical pharmaceutical sciences expertise, along with the medicinal chemistry and critical care medicine with Neomanoli, Lee, David, and myself, and developed a grant to evaluate these pathways. So we had developed a provisional patent, and to date we've screened over 150, I believe we're in 160s, new chemical entities that have been synthesized and screened. We developed a method for evaluating this in vitro, doing microzombial incubations to, again, make sure that the compounds either do or do not inhibit our intended target. Then we're able to evaluate first single concentration after the in silico prediction, the single concentration screening to be sure that our compounds inhibit, and evaluate in vitro to make sure they're effective inhibitors and selective inhibitors. This ultimately led to our in vivo evaluation, and we're working our way towards two lead candidates, and I will show you how far we've come with one of our two lead candidates to date in the research that we've conducted. So this work really has been led in many ways by Chenxiao Tang. Chenxiao is a PhD student in my laboratory, and I introduced Lingzhu Li earlier. Lingzhu Li finished her PhD. She's currently employed as a research scientist at Janssen Pharmaceuticals, and has been very effective in her career upon post-graduation from our lab. And Chenxiao has taken the work of the initial evaluation and really led to the screening and evaluation of the in vitro data that I'm going to be showing you, and she's approaching her graduation as we speak. So the initial step was screening the compounds. All of the 150 some odd compounds we've evaluated, we wanna be sure we have a potent inhibitor. So we have a way of evaluating in microsomal incubations and using our mass spec approach to be sure that it inhibits 20 heat formation. If it doesn't inhibit at least 50% at 500 nanomolar, then it's likely not potent enough for us to be interested. So that ends up weeding out of the 150 compounds, the ones we want to advance. Our compounds that inhibit at least 50% at 500 nanomolar, we evaluate for its potency, looking at 20 heat formation in vitro, and looking at the IC50 or the inhibitory concentration at 50% for each one of the compounds as our index of potency. Then evaluation of selectivity. I showed data earlier of eats versus 20 heat. We also look at the selectivity against all the other cytochrome P450s and a panel of six different probes for cytochrome P450. This is part of that clinical and translation as well, because we wanna be sure that any compound we develop is not going to have drug-drug interactions. So by screening in this early phase, we're not screening for all the possible drug-drug interactions, but we are screening for the major cytochrome P450s that could lead to complications in patients. And we want to avoid the advancement of compounds that will have that complication in the in vitro phase. So we don't find out that that's a complication when it's fully developed in the in vivo phase. We wanna be sure that the half-life is adequate, microsomal stability. So we put the compound in with the microsomes and see how quickly it disappears. How fast is it metabolized? This is a major limitation of many of the compounds that have been developed for this pathway. Another example of keeping the patient in mind is the solubility evaluation. Ultimately, we want to have a compound that could be administered intravenously as a neuroprotective early phase of injury. So we look at solubility and make sure we have good connect solubility for an IV formulation. So we don't have the formulation hurdle to be as high whenever we get to the drug development phase. And we also screen in vitro for blood-brain barrier permeability, is it a substrate for transporters? Because many times transporters are keeping things out of the brain and that would be a barrier for us being able to get our compounds into the brain to inhibit our pathway and our target. So hopefully this gives an example of that translational spectrum because we really are looking in vitro and thinking about the way this would be developed clinically so that we can remove some of the barriers of the drug development process along the way. Here's an example of some of our early compounds. This is our 20 heat formation rate. So if you have no inhibitor, no very low concentrations, you have 100% of 20 heat formed, it's not inhibited at all. And as you increase concentrations, you get this inhibition up to higher molar concentrations. And we're able to determine that 50%, what the inhibitory concentration is at 50%, this tells us how potent our compounds are. Our first compounds to hit our compounds UPMP10 and 19, they were around 400 and 200 nanomolar IC50 inhibition. And our other early compounds that were more potent were compounds 21 and 22, which gave us around 150 and 50 nanomolar. The goal here is really to have a nanomolar level inhibitor so that you have a very potent inhibitor and you're more likely to be able to evaluate the selectivity that you desire in the compound as well. So this is part of the selectivity data that we have and we have other selectivity data that I'm not showing today for interest of time. But what we were able to see here, again, this is percent of the activity, 20 heat is reduced, whereas the eats, which we do not wanna inhibit, were not affected by increasing concentrations. In this case, it was our compound 22 to show that we are getting selective inhibition of the pathway that we intend in vitro. But one of the problems with many of these compounds when they go in vivo and when they went clinically is that the biological half-lives were short. So we wanna make sure that we have good microsomal stability or good stability of the compounds. This is an example of a compound that was developed by Taisha Pharmaceuticals, TS24. And you could see at 30 minutes when it's incubated with microsomes, you only have around 30% of the compounds remaining. But the majority of our compounds we screen for how much of the compounds remaining at 30 minutes and at 60 minutes and see there's good metabolic stability, which should translate over into a better biological half-life as we move in vivo. So this is a summary of our lead compound to date, which we did take this compound in vivo. First, we do the screening, the potency, selectivity, microsomal stability, solubility, blood-brain barrier penetration, and compounds that make it through all of those criteria end up being a lead. We have a compound 107 that has a 50 nanomolar potency. It's 96.5% at 60 minutes. For microsomal stability, the selectivity window is around 500-fold with all the different P450s that we screen against the probes, not only the EATs. And it has good solubility. Again, for IV formulation, if this drug will ultimately be administered IV, it has good solubility. And it does have moderate blood-brain barrier penetration. It may be a substrate for PGP, but only at a moderate level. So it met our criteria for continuing for development. We very recently, and this is data that's very new, evaluated in UPMP107 at 20 milligrams per kilogram in the pediatric rat. This is not an injury. We have not approached injury yet. Those are the studies that are forthcoming. But in the pediatric rat, looked at the pharmacokinetics, initial pharmacokinetics, looking at concentration and peak of grams per mil over time and being sure that we are able to achieve brain concentrations of the compound in an adequate range to produce inhibition. And excitingly, we were able to demonstrate that UPMP did both get into the brain and also inhibited 20 heat formation the whole way out to nine hours after administration and below 50%. It was greater than 50% inhibition out the nine hours. Just to give you an idea, the HET16 compound inhibited, I believe it was 40% at three hours and then it was back to baseline. So it was a very transient inhibition with the HET16 compound and transient inhibition with other inhibitors. We were able to give higher concentrations and doses of the UPMP107 because of the selectivity. And we're able to maintain a good inhibition over out to nine hours to create a longer period of time for inhibition, which is also key. Even if we administer IV, it's important to have good half-life metabolic stability because the amount of compound that would need to be developed would be administered to patients as well, which is part of that target product profile for developing the drug compound. So a summary of this process has really led to a series of compounds. Basically, we have a leading series demonstrating selective human SIP inhibition. I wanna highlight one thing I didn't present today, but what was intriguing about our findings is that our compounds are actually selective for human over rat as well. So the compound 107 is actually more potent in human than it is in the rat cytochrome P450s because we're using the modeling to actually target the human enzyme. This is good and bad because it's good because it should be more effective in humans, and that's real goal, the end goal of the entire project. It's bad in the preclinical evaluations or critical to being able to figure out if the compound is effective before it can be advanced. So it is a bit of create some problems in the preclinical space and developing the optimal compound in patients. We demonstrated our compounds have improved metabolic stability, and we're really using the optimization characteristics in vivo to figure out what our best lead compounds are for pharmacokinetic assessment. And importantly, we're able to assess both the pharmacokinetics and the concentrations of the compound, but because we can measure 20 both in the preclinical animal models and also in human CSF and in patients, we have a way during the drug development process of knowing if the target was engaged. If the compound is able to get to the effect site and is able to reduce 20 heat concentrations, we'll know in vivo, theoretically, in preclinical we know now, in clinical we could know during drug development if the drug is indeed engaging the target that's intended for during the drug development process, which we believe would be very powerful in the success of compounds as they move forward. So hopefully this presentation gave a good example of the ways in which we've approached the translational research spectrum. Even when we're working in the basic science in vitro test tube space, or we're working in the preclinical research space, we have the clinical implementations in mind, the ways in which the drugs would be administered to the patients, the pathways, and if the pathways are relevant to the patient. And we have been able to conduct with our collaborators research to evaluate the clinical research to see if the same pathways that are being suggested to be important in the preclinical animal model are associated or correlated with outcomes in the clinical research that we have done. For this to be a perfect story, we'd be working in the exact same injury and the exact same insult, but by the nature of where the samples were and the ways in which we were able to collaborate. We've worked across subarachnoid hemorrhage, cardiac arrest, stroke, and various different models. But the theory behind the approach has been the same, which is to not operate only in one of these silos, but to really go between them and understand them and see ways in which we can collaborate to work across our different boundaries, bringing our expertise to the table along with our collaborators' expertise. And I wanna point out one other paper by Morgan in 2012, because this paper is an evaluation of success or failure in the pharmaceutical industry. And it was an evaluation of what they called the pillars of the probability of success to enter phase three clinical studies. So they retrospectively evaluated 44 phase two studies, and they looked for three pillars. First pillar was, in those phase two studies, did they know the exposure at the target site? So how much of the drug got to the target site over time, AUC at the target site from preclinical studies and or clinical studies? Pillar two was, there are some evidence that the target that they were intending to hit was occupied. Was there data in humans showing that they were binding to the target? So pillar one could be preclinical if it's tissue, because you can't measure the brain tissue target site. But if the brain tissue in the preclinical animal, is it related to the plasma AUC? And was there a plasma AUC that was targeted pharmacokinetically? Then moving to pillar two is, if you did get that adequate target occupancy, then is there data in humans showing that you were able to bind that target? What we talked about really here would, our case would be 20 heat concentrations in CSF. Would they go down? Could we use that as evidence that we're inhibiting the enzyme we intend to target? And then pillar three is evidence that modulation of that target would lead to a pharmacodynamic effect. In other words, you're going to protect, or you're going to protect the brain if you engage that target. If all three of these pillars were met in phase two, then eight out of the 14 studies entered phase three. If only the pharmacokinetics were known, only part of pillar one, then none of the 12 other studies made it to phase three. So this was interesting, would be interesting to add to this, to the current clinical trials to see the continued applicability. But the reason I wanted to highlight this paper is because the expertise within pharmacy is not sufficient in its own silo, but it's absolutely critical in the interdisciplinary drug development silo. There are so many studies I see, sorry, translational spectrum. There are so many studies that I see that are evaluating about drug dose, and they administer X dose into a preclinical animal model, and they're doing body surface area or body weight estimation of dose in human without knowing the exposure or the concentration relationship and not having a good handle on the actual target engagement. The pharmacology, the pharmacokinetics, the pharmacodynamics are our area of expertise within pharmacy that we bring to the table for the clinical and translational research development. And I would encourage any of you that are interested in those areas to take your expertise to them because it really is needed. The expertise within pharmacokinetics and dynamics are one of the major reasons, both repurposing drugs and new drugs have failed, one of the major reasons it failed in drug development. So this is my baseball analogy. You cannot hit a target that you can't see. So if we're not looking at the target, we're not going to hit the target. And are the basically having the right concentrations at the right levels and knowing that it's actually engaging the target both in preclinical studies and using that information to inform clinical studies will increase our likelihood of success and remove the blindfold when we're trying to find effective drug therapies. I have several acknowledgments and I'm happy to take any questions for the remaining time that we have. I've mentioned many of these people in the slides, Mia Minoli, Patrick Lohanick, Anthony Klein and our animal work and SAFRA Center for Resuscitation Research for the behavioral outcomes in the preclinical animal studies. Jeff Balzer has been instrumental in our subarachnoid hemorrhage patient studies along with our nursing colleagues, Beth Crago, Paula Sherwood and Mary Kerr who I mentioned in the slides earlier. My collaborators in pharmacy and chemistry of Lee McDermott, David Coase. My lab, Ling Zhu Li and Shenzhao Tang did much of the data that I showed today as well as Mark Donnelly who was instrumental in the clinical subarachnoid hemorrhage data. Trish Miller and Pat Oberle, your technicians in my laboratory have worked on these projects over the years as well. And finally, I'd like to support our NIH support. We have both an instrumentation grant for our mass spectroscopy equipment, the preclinical animal work R01s that have been funded to evaluate these projects and our current study, which is part of the NINDS IGNITE R21 and we're moving into the R33 phase of this grant for doing the development of the new drugs. With that, I would be happy to turn over the controls and take any questions that you may have about today's presentation and thank you all. Awesome. Thank you, Dr. Poliak for a great presentation. Just want to remind everyone, if you have any questions, you can use the question box at this point to ask those questions and I'll make sure they get relayed to our presenter. I guess while we wait on potential questions for the audience, can you tell us a little bit more about what you learned in your experience? And you talked in the beginning about breaking out of silos from a research perspective and collaboration-wise, you've built a pretty impressive research team. So how did you break out of those silos and build this research team that you have now? Thank you for that question. I really, when you see it 21 years into the career, it looks different than whenever it was year one and two. Keep that in mind for year five or year 10. And that's because these things build over time. They're individual relationships and it's really getting into a common environment where you can stop and talk about things that are common and relevant. In many cases, it was the journal club that we attended. And then once the relationship was established, it extended to our collaborative research. But there's that initial phase of determining what the interests are and then having meetings and following up and bringing the expertise together. We brought the pharmacy expertise to the Safra Center Group and the Safra Center Group brought their wealth of expertise in neurosciences. And really what I truly enjoyed about it is that there was respect for everybody and we were all genuinely interested in the same questions. And that really just spurns the collaboration. And it does take time, but it was not as if I sat in my office on day one of the job and wrote down all the people I wanted to collaborate with. So please, just to keep that in mind, that it is one of these processes that evolves and you develop the relationships with the key individuals and they grow, they recommend you to people, you end up building other people, you end up talking to other people through other people. It really is that genuine networking. Maybe tell us a little bit too, with the pandemic going on, changes, acute changes that have happened to research and how you think that's gonna impact you over the next maybe six months to a year going forward? Yes, I've said to my graduate students on many occasions, I'm gonna prevent predicting the future because so far every time I've done that, I've been wrong. So I'm going to avoid exploring too much about how we're moving forward. I can say it has been a challenging time in that the research laboratories have shut down for a period of about two months. However, we are back in the research laboratories and we are resuming our research in a thoughtful way with all of the parameters in place at the university. I think the biggest thing that the opportunity, those are the negative sides of the time that it takes to be able to do research and to be able to resume research. And that is even probably in some cases, even more impacted for clinical research and patient recruitment. However, the positive side of it is that not every meeting has to be an hour and meeting virtually is faster than going to everybody's office. Some things have to be face-to-face, but a lot can get done in a short period of time virtually. So I think if there's any positive to take their way, one of the positives from the changes in the process is I think that not everything has to be an hour-long meeting where everybody sees everybody, that you can get a lot accomplished in a short timeframe and you can get it accomplished with a large group of people. I've had the joy through SCCM of mentoring Dr. Hevner at the University of Maryland. And we had our first Zoom meeting face-to-face and it was kind of funny. We said we should have done this for the past two years. So I think that there in some ways you actually can improve your communications with individuals and actually increase the frequency, even though there are the disadvantages that I highlighted as well. All right, thank you very much. I'm not seeing any other questions from the audience. So we'll go ahead and conclude our Q&A session. Once again, I wanna thank you, Dr. Poliak. Thank you to our presenter. Thank you also to the audience for attending. Once again, you'll receive a follow-up email with a link to complete an evaluation. The link to that evaluation's also listed in the chat box for your convenience on the bottom of this page if you do not wish to wait for that follow-up email. Thank you. You only need to complete this once. Once again, there's no CME associated with this educational program. However, opinions and feedback are important as we plan and develop future educational offerings. The recording for this webcast will be available on the My SCCM website within five business days. And that concludes our presentation today. We wanna thank you all for joining us and take care. Thank you.

Models of Translational Research in Critical Care

translational research

collaboration

therapies for critical care patients

drug metabolizing enzymes

cytochrome P450 enzymes

20 HET metabolite

neuronal injury

target engagement