Dr. Shannon Cramm, Dr. Scott Fligor, and Dr. Savas Tsikis - Research Fellowship In Review

Today's a special day where we get to hear the kind of culmination of some of our own's work. And our first presenter today began her academic career at Michigan State, where she completed a degree in human biology and graduated with honors. This was her first foray into research through her work in a neurobiology lab studying the neuro basis of behavior. She then attended Michigan Medical School where under the mentorship of Dr. Engel Bay and Maggi, she developed a passion for health services research with projects and PDF transplantation. She's also completed a one-year fellowship in transplant epidemiology at Johns Hopkins with Dr. Sagev before beginning residency at Massachusetts General Hospital. During her time here at Boston Children, she's worked under the mentorship of Dr. Sean Ringel, completing her Masters in Public Health with specialization and clinical effectiveness at Harvard in 2021. It has been working on projects to optimize the management of children with appendicitis with publications and JAMA surgery, annals of surgery, JPS and Jax. For those of you who have worked with her clinically, like I have, you also know her as a kind, compassionate, and competent physician. Please give a warm welcome to Dr. Shannon Cramp. All right. Good morning, everyone. I'm excited to kick off our Grand Round Series on our research fellowships in review by talking about my work with the Eastern Pediatrics or DREAM Network to optimize the care of children with appendicitis. So my favorite non-essential organ needs no introduction to this group. Appendicitis is the most common abdominal surgical emergency of childhood and with it comes a significant burden and infections, healthcare utilization, cost, and antibiotic use. There's a lot of variation in how we care for these patients, not only in how we diagnose appendicitis, but how we manage it. And this results in variations and outcomes. This is likely due to the existing appendicitis literature, which is limited primarily in children to single-center studies with small sample sizes that have limited power and generalizability. The existing multi-center studies primarily leverage administrative data from FIS or NISQP pediatric data, which while is rigorously collected lacks the clinical granularity necessary to answer important management questions. And lastly, there's no standardization making it difficult to compare across studies in either the definitions of exposures or outcomes, but importantly, in how we adjust for appendicitis severity. This is where the Eastern Pediatric Surgery Network comes in. This is a multi-center research consortium established in 2019, and the collaboration comes together to share data, resources, and expertise. The membership votes on topics of high priority in pediatric surgical research, and appendicitis was chosen as one of the inaugural projects for the consortium. With their support, we created the UPSN appendicitis database. We started with NISQP pediatric data, because as I said, it's rigorously collected and arguably the gold standard and clinical data. It also has appendicitis specific variables. We augmented that data with dedicated chart review at each of the 16 participating centers, collecting things like operative report text, antibiotic utilization data, culture data, and laboratory data. With this data set in hand, we were able to ask a lot of interesting questions about the management of appendicitis, how to optimize antibiotic utilization, standardize severity assessment, what's the best intraoperative management, and defining the values or postoperative laboratory values. Now, we're limited in time today, so I'd like to focus on our projects in antibiotic utilization. And we looked at this through a lens of antimicrobial stewardship tackling this question systematically, thinking about when antibiotics are indicated, what is the minimum spectrum necessary, and the minimum duration necessary to achieve adequate treatment or prevention of infections. And so we'll start by talking about whether antibiotics are indicated in the postoperative period in children with gangrene as appendicitis. In one of my first projects as a research fellow, we looked at whether children with gangrene as appendicitis had increased risk of postoperative complications, and they had nearly twice the risk of infections. So can postoperative antibiotics mitigate this risk? To answer that, we looked at children with uncomplicated disease who had GSE findings, which we'd previously defined and validated, so gangrene, separation, or exudate directly involving the appendix. We excluded children whose postoperative length of say was greater than two days. And our exposure was whether or not any postoperative antibiotics were used. We looked at post-discharge outcomes of surgical site infections, abdominal imaging utilization, and revisit rates. We used propensity score matching to adjust for age, operative severity based on the presence or absence of gangrene specifically, and clinical severity for which we used postoperative length of say as a surrogate. We used mis-effects logistic regression to adjust for hospital level plustering of events. In total, we had 781 children with GSE appendicitis, of which 1.3% developed a surgical site infection. 53% of children received postoperative antibiotics, the median duration of which was two days. This graph shows that variation in the hospital level use of postoperative antibiotics in the blue bars, which range from 20% to 100%. The black diamonds represent each hospital's surgical site infection rate, which at most centers was zero and did not correlate with center's use of postoperative antibiotics. These were the results of our propensity score analysis. So we had 382 children between the groups that did not receive antibiotics, and that did. And our measures of patient characteristics and disease severity were well balanced between those groups. Looking at the table and forest plot, we see that the outcomes of surgical site infection, abdominal imaging, and revisits were no different whether children received postoperative antibiotics. So from these data, we see their significant variation in the management of postoperative antibiotics in GSE appendicitis. And overall, the risk of surgical site infection is extremely low and not associated with postoperative antibiotic use. So these antibiotics may not be indicated in children with GSE appendicitis. Next, we'll look at antibiotic spectrum, and we'll shift our focus to children with complicated disease. Do these children benefit from impure abuse of anti-suitomonal antibiotics? And so for this cohort, we looked at children with complicated disease using a previously defined and validated definition from two prior ringell fellows, Dr. Seema Nandoar and Daniel Cameron. We excluded those whose postoperative length of stay or antibiotic duration was fewer than two days. And we compared children who received Zosin or Septraaxone with metronidazol, which represent the two most common antibiotics used in complicated disease, as well as the most common antibiotics that do and do not cover pseudomonas respectively. Our outcomes were postoperative drainage of organ space infections and their resultant culture data. Again, we used propensity score matching, this time to balance on the number and relative distribution of intraoperative criteria of complicated disease, which are listed in the orange box. And again, use mixed effects logistic regression to adjust for hospital level clustering. This bar graph shows the variation at the hospital level of antibiotic regimens used in these children with light gray representing Septraaxone metronidazol and black Zosin. The diamonds represent the hospital level drainage rates. As you see, there's a lot of variation in the spectrum of antibiotic used across the centers in our study. And there's no association between the drainage rates and their use of an anti-stutomonal agent of Zosin. These are the results of our propensity match analysis, which included 782 children between the pipersonal and vasobactam and Septraaxone metronidazol group. After matching, there were no differences in disease severity between these two groups. The first line of our table looks at the risk of having any drainage procedure for an organ space infection, which was no different between the antibiotic groups. Each subsequent line looks at the different risk of growth of any microbe, E. coli, and various other microbes. We see that the risk of having a drainage procedure that results in any microbe growth was no different. However, the risk of E. coli was actually higher in children treated with Zosin. Importantly, pseudomonas was no different between these two groups. The issue of the results of all 104 cultures obtained from children treated with either pipersonal and vasobactam in medium blue, or Septraaxone flagel in light blue. This is no longer the propensity match analysis, but rather represents all cultures obtained. If you look at whether or not any organism was isolated, there was no difference between the two antibiotic groups, although Zosin had a isolation rate of 63% where a Septraaxone metronidazol 45%. Similar to the propensity match analysis, we see that the isolation of E. coli was higher from cultures in children treated with Zosin than Septraaxone metronidazol. The all-important pseudomonas was no different between the two antibiotic groups. From these data, we again see significant variation in the empiric spectrum of antibiotics used in children with complicated appendicitis. An anti-sutomonal antibiotic use of Zosin was not associated with reduction in either drainage procedures or ultimately the growth of pseudomonas. These data would support limiting empiric use of anti-sutomonal antibiotics like Zosin and children with complicated disease. Next we'll look at antibiotic duration. What is the ideal post-operative duration of antibiotics in children with complicated appendicitis? So again, using a cohort of children with complicated disease, this time we excluded those who had an organ-space infection diagnosed during their index day or had an empiric post-operative antibiotic regimen greater than 14 days. We defined the empiric antibiotic duration as the number of post-operative antibiotics given during the index hospitalization and prescribed at the time of discharge. And we used quartiles to describe our duration groups to reflect contemporary practice. Our short duration included the first quartile and the long duration included quartiles two through four. Our post-discharge outcomes were organ-space infection, treatment failure, abdominal imaging, and revisit rates. Again, we used propensity score matching based on patient characteristics, disease severity, and clinical severity, and used mixed effects regression to adjust for hospital level clustering. We had nearly 1,300 children with complicated appendicitis and on the left, you see the hospital level data in a box and whiskers plot showing the antibiotic duration, which ranged from the median of three to 11 across the centers. On the right is the histogram of all patients included in the cohort. You see our short duration or the first quartile is children who received five or fewer days of antibiotics and the long duration is children who received six to 14. These are the results of the propensity match analysis, which included 622 children in the matched cohort. Again, after matching, there was no different in-patient patient characteristics or disease severity between the short and long antibiotic duration groups. If we look at the table and the forest plot, we see there was no difference in our outcomes between those who got five or fewer days versus six to 14 days of post-operative antibiotics. Again, here we see that post-operative duration of antibiotics varies widely across hospitals. The antibiotic treatment duration that exceeds five post-operative days was not associated with better outcomes and may ultimately be unnecessary and children with complicated disease. With all of this variation that we've seen in antibiotic management, we began to wonder, can we leverage the principles of positive deviants to identify exemplar hospitals and potential opportunities for quality improvement? In other words, if we're all doing something different, is someone out there doing it better. So this was a proofing concept analysis in which we looked at the 16 hospitals in the EPSN, limited to their populations of children with complicated appendicitis. And we looked at their hospital level outcomes as balancing measures, organ space infection rates, as well as their cumulative antibiotic treatment duration. This time defined as all days of post-operative antibiotics in the 30-day post-operative period. So including index hospitalization, days prescribed at discharge, and days prescribed after discharge, whether that was as an outpatient or at a readmission. We calculated observed two expected ratios for each hospital using multi-level mixed effects regression to adjust for hospital's patient characteristics, as well as disease severity case mix. These are results of this balancing measures report, which we had nearly 1,800 children with complicated appendicitis representing a median of 109 patients for hospital. The organ space infection rate overall was 16 percent with a median cumulative antibiotic utilization of nine days. 50 percent of hospitals were outliers for antibiotic utilization and 19 percent for organ space infection. Example our hospitals are highlighted in our box in our scatter plot. Sorry, let me move that. Our highlighted in this scatter plot where each point represents, oh, that just did it on my screen. Anyways, that represents an individual hospital. The blue represent exemplar hospitals who had lower than expected either organ space infection rates on the Y-X-axis or antibiotic utilization on the Y-axis. And those highlighted in orange represent those who had opportunities for quality improvement or higher than expected organ space infections antibiotic utilization or both. From these data, we see that there is significant variation in cumulative antibiotic utilization as well as outcomes among hospitals. And a report card, such as this, was able to adequately discriminate performance outliers. So a report based on balancing measures could be used for comparative performance benchmarking to identify opportunities for quality improvement as well as opportunities for best practices. So taking a step back as we think about the lessons we've learned from the EPSN appendicitis database, I think that there's a lot of variation that we've seen in how antibiotics are managed in children with appendicitis and that more intensive antibiotic utilization does not equate to better outcomes. And so there's significant opportunities for antimicrobial stewardship in the management of children with appendicitis. And so with that, I'd like to thank my mentor, Sean Ringell, who's been an extraordinary teacher over these past three years. I'm really grateful to my co-fellow, Dr. Katherine He, our best statistician, Dr. Deon Graham, as well as our collaborators here at Boston Children's Hospital and our EPSN member hospitals. I'd also like to thank all of you, the faculty, the fellows, my research co-fellows, as well as the NPs and residents who have worked with me over the past three years. I have grown so much as a researcher, as a clinician, and a surgeon. Thank you. That was really spectacular. We're going to have all three and then take questions at the end just to make sure there's time. Our next speaker is a Massachusetts native from just down the street in Newton. He completed his undergraduate degree at Georgetown University, where he graduated cum laude with a degree in biology of global health. From there, he acquired his medical degree from Virginia Tech, as well as medicine as a member of AOA. He then transitioned to surgical residency at the Bethescher old Deaconess Medical Center and started his research fellowship here at Boston Children's in 2020. Primarily working under the tutelage of Dr. Mark Puder, Dr. Flegor has multiple academic and research awards, including outstanding resident teaching award from Harvard Medical School, Chairman's Fellowship from Boston Children's Hospital, and the Harry M. Barr's award presented to an early career investigator for the top resident paper at Aspen. He currently has a pending patent. He has over 20 peer-reviewed publications and multiple national presentations. It's with great pleasure that I introduce him. Today, he will be talking about the preclinical development of a new therapeutic for intestinal failure associated liver disease. All right. Okay, thank you for the kind introduction. I'm excited to be presenting some of our work today, focusing on the preclinical development of a new therapeutic for intestinal failure associated liver disease. And this work was funded by Norseath Air Putics. So intestinal failure is the inability to absorb adequate nutrition through the gut due to inadequate bowel length or function, and it requires parental nutrition to support growth and development. Typically, in children, we see this in the setting of neonatal gastrointestinal emergencies requiring substantial bowel resection. Intestinal failure associated liver disease is a key life limiting complication of long-term parental nutrition use and intestinal failure, starting with an active phase of colostasis and inflammation, and progressing to a chronic phase of hyprosis and stiotosis. And while modern management has essentially turned this from early catastrophe, early in life, requiring liver transplant or resulting in death to more of a chronic, smoldering liver disease, this is something that's highly prevalent in our intestinal failure populations. And two-thirds of children on a routine liver biopsy will have abnormal liver histology and publication recently from Greg Keefe here in Boston Children's found that almost all of our patients who are on long-term parental nutrition have abnormal transaminases. So the long-term implications of this smoldering liver disease are unclear, but every year we have more and more kids who are living years or even decades. So it's critical to discover new therapies to prevent and treat the prevent and treat eye-fowl. So some of the work I'm going to be presenting today is on the development of CFA-6179, is this candidate therapeutic for intestinal failure associated liver disease. And I'll discuss some of the key proof of concept studies that we conducted here at Boston Children's, which has helped propel this drug from early mechanistic data into human clinical trials. So fatty acids are intriguing therapeutics for metabolic and inflammatory liver diseases, but generally they're rapidly metabolized for use as a fast source, they're incorporated in cell membranes, and they have a very short half-life, they're very weak agonists of their target receptors, which makes it so in an unmodified form, they are not necessarily going to be effective therapeutic. CFA-6179 is a structurally modified medium chain fatty acid analog. It's based off a dekinobic acid backbone that's absorbed directly into the portal circulation. And it avoids bidoxidation and cellular membrane incorporation, which allows it to achieve the necessary in vivo concentrations to really hit some of those key receptors. And in vitro, we know that it's a GPR-84 partial agonist, a PPA-R alpha partial agonist, and a PPA-R gamma full agonist, which is a receptor profile that would be expected to have broad metabolic and inflammatory and and anti-phibrodic effects. And there was some extensive urine data before we got to this drug that was looking at CFA-6179 in various metabolic liver diseases. And some of this data includes improving insulin sensitivity and glycemic control in a type 2 diabetes model, and a lot of work in a non-apaholic scale hepatitis model demonstrating decreased liver enzymes, improved seotosis, anti-phibrodic effects. But we then wanted to take this potentially promising drug that could be a good therapeutic for eye-fowl to evaluate it. First in our standard murine model of intestinal failure associated liver disease, where mice are given an oral adlib, high carbohydrate diet that's fat-free, and every other day tail vein injection of one of the available lipid emulsions. And what we found was that mice that received introlipid in this model with a medium chain triglyceride vehicle treatment developed fatty liver and biochemical abnormalities. But when we treated with this drug, CFA-6179, we maintained normal liver histology without fat and maintained normal liver enzymes. And on a lipidomics analysis, we found that CFA-6179 decreased pro-inflammatory lipid metabolites, such as a racodonic acid derivatives, compared to the vehicle control, as well as these key mediators of lipotoxicity, which is the damage that occurs to the liver after fat overload, and they're decreased diacyl glycerols and ceramides. And this may be important for minimizing the damage that occurs from a fatty liver. So there's all of this preclinical mouse data, but in order to fully decide to develop this drug and fund this drug, we need to proof of concept in a large animal model. And the best large animal model for eye-fowl is the preterm piglet model. And in this model, piglets are delivered preterm. They're given parental nutrition for two weeks, after which they develop histologic and biochemical markers that parallel what we see in humans. So we obtained pregnant Yorkshire South, and we delivered piglets five days preterm via cesarean section. Following resuscitation, we implanted regular central venous catheters. Piglets were started on parental nutrition, and this was continued as the sole source of nutrition for 14 days. Piglets were randomized to receive treatment with either CFA-6179 or a medium-chain triglyceride vehicle via daily or gastric gavage. Blood was drawn at day of life 1, 8, and 15 to assess biochemical markers. Our primary outcome was biochemical colostasis, and secondary outcomes included histologic and transcriptomic endpoints. Piglets in the MCT vehicle group developed a direct hyperbillarubinemia with a direct billarubin of 1.6 and a total billarubin of 2.6 milligrams per desoliter by day of light 15, treatment with CFA-6179 prevented biochemical colostasis. Other markers of colostasis were assessed including gamoglutamil trans race, a marker of kalansioside injury, and plasma bile acids, and these increased throughout the 15 days in the MCT vehicle group, but CFA-6179 had significantly lower GGT and bile acids at day of life 15. We also assessed histologic markers of intestinal failure associated liver disease, including seotosis, which was assessed two ways, first with an oil red O stain, which stains fat red, representative histology is shown on left, and there was decreased oil red O staining in the CFA-6179 group, as well as decreased tissue triglyceride content. So CFA-6179 prevented seotosis. We performed a cytokaritin-steven stain, which stains colandiosides to assess bile duct proliferation, and piglets in the MCT vehicle group had extensive bile duct proliferation, and this was reduced by CFA-6179 treatment. We also assessed hepatic phybrose or hepax cell activation, which is a key step in fibrogenesis using an alpha-SMA stain, and there is decreased alpha-SMA staining in the CFA-6179 group, and we had a masked pathologist assessed fibrosis using the ISHAC fibrosis score, and there is a median score of three in the MCT group, which is fibrosexpansion of most areas with occasional portal-portal bridging compared to a median score of one in the CFA group, which is just fibrosexpansion of some portal areas without bridging. We also performed a transcriptomic analysis and found that CFA-6179 treatment broadly increased betaoxidation and fatty acid metabolism, while having broad anti-inflammatory and anti-phibotic effects, including inhibition of NFCAPA-V signaling and hepatic fibrosis signaling mediated by TGF beta. So in this preterm piglet model that's highly analogous to the human neonatal eye feld, we found that CFA-6179 prevented colostasis and seotosis and reduced bile duct proliferation and fibrosis, and these data were allowed the company to make the decision to fully fund this drug and move forwards with formal admiand IND-enabling studies, so the formal preclinical toxicology studies that allows for the submission of investigational new drug application. So that's all very exciting. CFA-6179 looks great in this preterm piglet model, but the next question we had is, is entral administration even feasible for the intestinal failure population? As we all know, most of our children who are receiving long-term parental nutrition have substantial bowel loss and may not be able to adequately absorb this drug to make it effective. So our next step was we performed a mini pig study to evaluate the absorption of CFA-6179 in short bowel syndrome. And short bowel syndrome is heterogeneous disease in terms of the remnant intestinal length, the anatomy of the reconstruction, and the motility and function of the remnant segments. And safety if drug absorption affects the safety and efficacy of a drug, and there's some older research from Piagepicin that showed that in humans with short bowel syndrome, absorption of medium chain fatty acids depends upon the presence or absence of a colon, as children who have their colon in continuity will absorb almost all of the medium chain fatty acids you give as opposed to children who do not have an aneroctomy. So it was critical for us to understand whether this drug could feasibly be administered through an enteral route to this population. So we obtained five-month-old female eucotain mini pigs. These are young adult mini pigs, and we sought to have data that was most translatable to the young adult population as the initial phase two study is going to be conducted in adults. And we decided upon two models of short bowel syndrome to evaluate the spectrum of absorption that we may see in our patients. The first is an established Genoelial Anastomosis Model. This is type three SPS, where we resected 90% of the Genoelial Length and created a Genoelial Anastomosis with five to one length, Ramnand Jajunum to Ilium. The second model is a new model that we created for this study, which we've termed as pseudo-Jajunus to me, where the proximal Jajunum is attached to the retroperitoneal colon immediately prior to the rectum with bypass of the intraperitoneal colon, which is nearly all of the colonic length in a pig, as well as the Iliocecal valve, which essentially creates or replicates the physiology of the proximal enorostomy. Five days prior to bowel resection, when the mini pigs had the entire entirety of their bowel, we performed a single dose pharmacokinetic study with a single dose administration of C5-6179 via oragastercavage. We evaluated two formulations, aqueous and non-aqueous, and performed serial blood draws over 24 hours. After a washout period, we performed a 90% bowel resection and allocated piglets to either the Genoelial or the pseudo-Jajunus to me group. And then on post-operative day three, we performed a second single dose pharmacokinetic study with five pigs per formulation and per bowel anatomy group for a total of 20 mini pigs. And to orient you with these absorption plots, we assessed the total absorption of the drug using area under the curve and normal bowel absorption. So pre-resection is in black, 90% Genoelial resection with the Genoelial and Asimosis is in orange, and the 90% resection with pseudo-Jajunus is blue, and absorption is also subdivided by formulation. We found that compared to piglets that had the entire complement of their bowel, the normal bowel piglets, there was 36% decreased absorption in the Genoelial group, and compared to normal bowel, there was 74% decreased absorption in the pseudo-Jajunus to me group. And so this tells us that massive small bowel resection does affect the absorption of C for 6179. And we also sought to determine the effect of bowel anatomy and found that looking at the piglets that had 90% small bowel removed, the piglets that had the pseudo-Jajunus to me as compared to the Genoelial group had 41% decreased absorption. So bowel length and bowel anatomy affect absorption of this drug. Finally, we evaluated the effect of the two different formulations, the non-aquias, the non-aquias formulation, which is in medium-chay triglycerides, or the aqueous formulation, which is the saline suspension, and found that the aqueous formulation had 55% decreased absorption. So the non-aquias formulation was selected for further clinical development. So to summarize this pre-clinical body of work, C for 6179 is effective in urine models of gnash and eye feld. And it's effective in a pre-term piglet model of eye feld that closely that's closely analogous, highly analogous to the human neonate. And these data were included in the IND filing package and were the basis for advancing to phase one trial. And then finally, we evaluated the absorption of C for 6179, finding that it was affected by a testinal length in testinal anatomy and drug formulation. And the design of the upcoming phase two trial was changed due to this work. And the groups of patients include in this study will be balanced by intestinal anatomy, that is whether or not they have an enorostomy due to this differential absorption that we noted. And in addition, the non-aquias formulation was selected for the clinical trials due to improved absorption in the mini-pigs. So where has C for 6179 gone? So it just completed a phase one trial in the last couple of months. And this work was just presented at Aspen. There were no severe adverse events and no issues. So it has now moved on to phase two clinical trial, which will start in the fall. The initial phase two study will be performed in adults. We are looking at the feasibility of including Boston Children's as a possible site because we do have a number of patients who are over 18 years. But there are a number of confirmed sites already that have large short bowel populations. And we're currently planning to connect a pediatric phase to a 3-trial at Boston Children's. So I'd like to say thank you to the department for supporting me for the last three years and helping me develop. I focus on all the members of my lab, Dr. Seges and Dr. Hirsch, my co-fellows. Kathleen Gura has been a great help in all of our federal nutrition and lipid projects and couldn't have done those without her. Other members of our lab, Amy Paine, our research technologist, research assistants, Michaela and Sarah, Paul Mitchell, our biostat station. And then I also have some other fellows from other labs who jumped into the trenches with us with the pigs, Camilla Moscaozova, Ashlyn Whitlock, Lorraine Arinkone, Cruz. And finally, I'd like to thank my mentor, Dr. Puder. I've really developed a lot over the last three years as a surgeon, scientist, and had a really robust experience with translational medicine and drug development. I think the opportunities I've had in this lab have been pretty unique and they're not something that you can find anywhere. So thank you very much. I guess I'll take questions later. Finally, I'm happy to introduce Dr. Savasikis, born and Princeton, New Jersey, which I believe is south of here. Most things are south of Boston, but I'm not great on East Coast geography yet. He completed his Bachelor of Science in Molecular and Cellular Biology at the University of Connecticut, graduating summa cum laude with honors. He then traveled to the University of Chicago, where he completed his medical degree, and then transitioned to surgical training at Beth Israel. And finally, started his research fellowship here in 2020 under PI Mark Puder. Savas is a member of the Gold Humanism Honor Society and his co-author on a Phase 3 Open Label Grant, which is related to the topic he'll be discussing with us today. He has over 20 peer reviewed publications and close to 40 presentations at professional meetings. Today we'll be learning about the work he's done on the Digestive Cultures known as ReloSorb and its potential as a therapeutic strategy in pediatric shore bowel syndrome. Thank you, Patrick, for that wonderful introduction. Today the topic of today's talk is for bowel syndrome harnessing the power of his gut. And today's talk I will provide an overview of the rationale, background, pre-clinical studies, and then finally human clinical trials for the Digestive Cultures commercially known as ReloSorb in the use of pediatric shore bowel syndrome. Through my three years as a resource fellow, I've had the opportunity to see an idea be generated in the lab translated into late pre-clinical animal models and then eventually make its way into humans. And I hope to share key elements with that journey with you today. As an initial disclosure, the presented work wasn't part supported by AcreStheraputics, which is the manufacturer of the device using these experiments. As we are all familiar with, shore bowel syndrome occurs as a result of loss of intestinal functional area resulting in intestinal failure. Intestinal failure is characterized by nutrient malabestorpsin, dependence on intravenous parental nutrition, and can occur in both children and adults. Causes range from congenital, such as gastro-stasis, intestinal entresia, malrotation with ovulus, to acquired causes with an infant's nectrotizing endocolitis being the most common, and in adults trauma, radiation, grown disease, ischemia can all contribute to SPS. While long-term use of parental nutrition is life-saving for these patients, long-term use is associated with several important life limiting complications. These can include sepsis from catheter-align associated bloodstream infections and a disease entity that Scott just mentioned, which is known as intestinal failure associated liver disease. A key element of SPS to understand is that survival, in part, depends on being able to wean from PN by achieving enteral autonomy. Patients with SPS also suffer from several key gastrointestinal issues. These include chronic diarrhea from fat, protein, carbohydrate malabestorpsin, intestinal dysmatility, small intestinal bacterial overgrowth, and key nutritional deficiencies. The issue of nutritional deficiencies is highlighted by a retrospective cohort study by Young et al. In this study, the authors examined the incidence of key nutritional deficiencies as patients transitioned from PN2N and after achieving enteral autonomy. As highlighted in these graphs, the incidence of key fat-soluble vitamin deficiencies increases several foes after achieving enteral autonomy. These findings support the, highlight the need for continued observation of these patients, and also the potential issues that can arise from these nutritional deficiencies, which can include metabolic bone disease. Google-like peptide of GLP2 is an important intestinal growth factor in SPS. It is produced by Entera Endocrine L cells of the Ilium and Colin and is known to induce intestinal adaptation through its positive intestinal terropic effects. In fact, Teteroglutide, which is a GLP2 analogue, has been shown to promote intestinal adaptation absorption with benefits on weaning and intravenous fluid support in pediatric SPS. While Teteroglutide has revolutionized the approach to and has become an important part of the toolkit for the management of patients with sore bowel syndrome, many patients still fail to advance on their enteral nutrition, as nutritional male absorption and fat male absorption in particular can become key limiting factors on achieving enteral autonomy. To better understand fat male absorption in SPS, an overview of intestinal fat digested and absorption is provided. Fast, enter the intestinous globules that are then acted on by bile salts through the process of emulsification. Light paste released by the pancreas can enact on these emulsant droplets, breaking them down, breaking down the fats into their monoglyceride and free fatty acid form, that can then form micelles with more bile salts and are absorbed across the intestinal and pithylilare into the lacteal system. In stroke bowel syndrome, the combination of rapid intestinal transit resulting from intestinal dysmetility and decreased intestinal length means that by the time these processes and reactions have occurred, the fats have already entered either the ostomy back or the colon, resulting in male absorption of fats, fat soluble vitamins, and potentially continued dependence on pancreas nutrition and an inability to achieve enteral autonomy. We hypothesized that by bypassing these key initials great limiting steps through the extracorporeal digestion of fats, you may be able to accelerate fat digestion thereby reducing the need for parental nutrition support. To evaluate this hypothesis, we utilized the immobilized life as cartridge, which is commercially known as realizorb, and this cartridge is already FDA approved and currently in use in patients with cystic fibrosis who have extra-cream pancreatic insufficiency and are maintained on enteral nutrition. The cartridge contains immobilized leaves of lipase and connects in line with existing enteral feeding sets. As fats in enteral formula passes through the cartridge, the beads with contained lipase act on them, hydrolysing the fats and breaking them down into their monoglyceride and free fatty acid forms. Thereby allowing broken down fats to be directly delivered to the patients. This would bypass the initial key limiting steps that I mentioned earlier when discussing the pathophysiology of fat absorption. It also avoids the issue that exists in pre-digested elemental formulas as pre-digested fats when sitting in an enteral formula feeding back can become rancid through the process of lipid peroxidation. We therefore hypothesized that pre-hydraulic fat and enteral formula will reduce fat malabsorption in SPS. To evaluate this hypothesis, we designed and executed an initial pre-clinical study in which Mel-Yorkstar piglets had SPS surgically induced. Three groups were studied on control group that underwent no surgical resection, and 275% distal intestinal resection groups, which had SPS surgically induced. They only deferred on whether or not they received enteral feeds through the use of the mobilized lipase cartridge or ILC. Animals had simultaneous placement of central lines for the provision of fluid and antibiotics and gastrocymetubes, and received continuous enteral feeds throughout the study. They also add access to oral pig chow at 40% of their gold calories, and no pn was used in this initial experiment. The animals were studied for 14 days, and the primary outcome is the coefficient of fat absorption. The coefficient of fat absorption is a metric of the percent of intestinal fat that gets absorbed across the intestinal layer. It arises from a 72-hour process that involves detailed nutritional intake of all input, which includes enteral nutrition, as well as oral intake, and a 72-hour stool collection was once performed in the pigs, and the total fat is then quattified in the stool. The percent CFA is essentially the percent of fat that gets absorbed. We demonstrated that ILCUs improved fat amount absorption in this preclinical model, as demonstrated by the lack of a difference between the sham and the 75% resection group that received the feeds with a digestive cartridge. In contrast, there was a significant difference in the percent CFA, and animals that did not receive the cartridge. ILCUs was also interestingly associated with increased plasma concentration of key fat soluble vitamins D and E. Based on these initial preclinical results, we then hypothesized that if fat amount of absorption is improved, you may also reduce the dependence on parent-trail nutrition. To evaluate the hypothesis, we designed and executed a second preclinical study, and with, again, 5-week-old male York's health piglets had SPS surgery clean-duced through 75% to soldier-journal aloeilary resection, but the key difference here is that, most operatively, animals were maintained on parent-trail nutrition. After recovery, EN was initiated, and two groups were studied, a control group and a treatment group that deferred only on whether or not they received feeds with the ILC. Animals were studied for 14 days, and the primary outcome in this study was the change in PN and EN calories. This is a schematic representation of the preclinical study. Animals had SPS surgically induced a study day minus four. PN was initiated at 25% of goal calories, and then advanced by 25% each day to ensure adequate tolerance until receiving a 100% of goal calories a study day minus one. Then at study day one, PN was decreased by 20% in both groups, and EN was initiated at 20% of goal calories. Animals were eligible for further advancement of their entrial nutrition every other day for the 14-day study if they fulfilled certain clinical advancement criteria. These included the absence of DERIA and the presence of at least 100 gram per day weight gain. Labs were drawn at baseline, which was defined as study day one, prior to the initiation of entrial feeds at study day eight, and then at the end of the study of study day 15. All labs were fasting, and lab draws to ensure that entrial nutrition did not, the provision of either EN or PN did not affect the immediate lab results. Overall, use of the LC was well tolerated, and there were no significant adverse events observed in either the control or treatment groups. There were no significant elevation in either liver enzymes for total bilirubin, and they remained within normal limits throughout the whole study period. Lineded, histopathologic evaluation by a veterinary pathologist also demonstrated in no significant evidence of systemic toxicity in either liver tissue, slenic tissue, or kidney tissue. In terms of the primary outcome, I'll see animals demonstrated significant reduction in their overall PN dependence over the course of the 14-day study. By the end of the study, there was a 19% difference between control and treatment animals with a 95% confidence interval between 15 to 24%, which is both statistically and clinically significant. The converse of this graph is the increase in the end advancement that was observed over the study, with treatment animals demonstrating an average rise of 66% in their entrial nutrition, compared to just 47% for the controls. Flasmagial P2, which is known to induce intestinal adaptation, was also significantly elevated by the end of the study in ILC animals compared to controls. This correlated with an impressive increase in intestinal length by almost 20% in treated animals, compared to just 1% in the controls. To determine if these findings correlated with increased intestinal cell proliferation, immunostaining for the proliferation marker, Ki67 was performed. As demonstrated by representative immunofluorescence sections on the screen, Ki67 staining, which is marked by green, was higher in treatment animals compared to controls, and this was confirmed on quantified analysis of the average number of Ki positive cells per crypt, which was twofold higher in treated animals compared to controls. ILC use also resulted in significant elevation of key omega-3 fatty acids, including DHA, EPA, and the overall concentration of omega-3 fatty acids. These findings persisted even after adjusting for the percentage in, as there was still a significant difference confirming that ILC use resulted in an increase absorption of these fatty acids. ILC treatment resulted in higher plasma nutritional markers at day 15, which included vitamin A, HDL, and LDL. Based on these two preclinical studies, we can conclude that ILC use in conjunction with antropheating, reduced pN dependence, improved nutrient absorption, and increased bowel growth and adaptation in this preclinical model. PN-related toxicity is an important complication with significant morbidity and mortality in SPS, and based on these results, we can hypothesize that ILC may be an alternative therapeutic for the pediatric SPS population. Based on these results, we designed and obtained funding for clinical study to study the commercial relies work used in pediatric SPS. We received an FDR-1 fund, and the study is a 90-day Phase III open labeled exploratory study with a pre and post-study design in which each subject serves as their own control. Like in the second phase preclinical study, the primary outcome is a change from baseline and PN calories, with secondary outcomes being the change from baseline and body weight for with with with a with adjusted weight for HD scores. Patients with SPS who are aged as two to 18 years of age are eligible for inclusion, and a key element for inclusion is the stability on their on both PN and EN prior to enrollment, defined as less than 5% reduction in PN for one month prior to screening, and with and in terms of entral nutrition, defined as no change in formula composition or rate for one month prior to screening. Given the pre and post study design, we need to ensure that patients any changes in their entral nutrition regimen are observed as a result of the device and not necessarily because of the natural history of their disease process. With that in mind, patients who have initiated tethered blue tide, which is known to promote intestinal adaptation within the past six months of screening are excluded. However, after six months given stability, they can be eligible for inclusion in the study. This is a schedule or events of the study, just to highlight that it's a particularly complicated study, and key features include a daily study coordinate phone call for adverse event monitoring, as recommended by the FDA, a 70 to R FIQO collection to calculate the coefficient of that absorption, which is done both at baseline and at the end of the study, and daily nutritional intake as we want to keep a close eye on the nutritional intake of these patients. We've already enrolled three patients and these three patients have recently completed the trial. We did have an issue with the 2022 formula shortage, which impacted initial enrollment. However, we do anticipate that we should be able to hit our target enrollment of 32 patients over the next four years. At the same time, as the clinical 90 day FDA funded study was started, we designed and obtained IRB approval for an extension trial, which is designed for participants who have completed the original study in order to provide additional time points to assess these various key outcomes. This study is fully funded by our press of therapeutics, and so far, all patients who have been in the original trial have agreed to participate in the extension trial. And if we have a similar target enrollment of 32 patients over the next four years. Overall, both trials will provide important initial preclinical data to assess the role of Eliza within pediatric SDS, and this brings us to the third step of taking something from the labs to the patients, and I look forward to the continued results of this of this device and being a participant in this process. I want to thank everyone in our study team, who's with without their contributions, that each studies would not have been possible. This include my co-fellar, Scott Flurger, Thomas Hearst, Amy Can, Paul Mitchell, our bio-satisfaction, and of course, Kathy Gura, our pharmacist who expertise and input would not not without her expertise and input, we would not have been able to conduct these studies. And there's there's a force and an important need to thank my mentor, Dr. Mark Peter. I've never had a mentor who's been so available and committed to our success, and without his help, guidance, assistance, and presence, we would not have been possible to perform these studies. We should acknowledge our funding sources, which include an R1 grant from the Food and Drug Administration, and T32 grants from the National Institute of Health, Acreta Therapeutics, the BASCO Biology Program, and of course, the Department of Surgery at Boston Children's Hospital. And with that, I'll be happy to take any questions with the rest of our headquarters. Thank you very much. We'd like to open up the questions to Shannon, Scott, and Savas, who just did a wonderful job presenting their fantastic work. I was wondering if the mentors would like to make a comment first. Thank you, Scott. Savas, I want to start out with a little bit of an apology because the amount of work that was put into this was extreme. I mean, 24-hour shifts exhaustion, and I've never apologized for that before, but it was extremely intense. With the hundreds of Balvar sections, the 24-hour care of the pigs and the piglets. This is just part of what these two have done, and they've also worked on developing other drugs and devices. They've worked with the patients very closely, the NIH, the FDA, and companies, and to the point where the companies want to have them stay on as advisors for two separate companies, and plus the new company that's been formed around new lipid emulsions. And lastly, these two are responsible for bringing over $6 billion to the research lab. Thank you very much, both of you. Great. Well, I think that today, it's demonstrated that 15 minutes is not enough for most of our fellows, and I say that for all three, and some really incredible work. For Shannon, congratulations on really three years of just incredible work. And I think that you've seen some of the academic productivity that's pretty clear, but I think what is really understated in the presentation was the first third of those slides with building this consortium, having this vision of really developing a set of projects that made a lot of sense in trying to get people to change their practice. And the organization and leadership skills that Shannon had with bringing this to fruition was you could probably set up a one hour discussion just on that. And what many people don't know is that within the consortium, Shannon was invited to give two grand rounds at consortium hospitals. Not me, Shannon. And that's because of the vision, the leadership, the organizational skills. And I think that really is what's in addition to having the academic acumen really as the true mark of a future academic leader in health services research. So it's been an incredible opportunity. It's been like driving in an Uber car in the back seat, texting, well, you get to where you need to go. And so congratulations. And you should really be proud of what you accomplished. So opening up to questions for all three of the folks. And if they wouldn't mind just going up to the front. And we have a little bit of a slave between the official clock here and the actual time. So we have a few minutes. Well, since there seem to be no questions, I have questions. First, Shannon, for you, you know, each of your studies shows a bell shaped curve, which is sort of the fundamental variable in biology. And I was wondering, do the hospitals that seem to have the issues with antibiotics in your studies? Do they stay the same? Or are they just different hospitals at different times? There is certainly a group of hospitals that favor intensive antibiotic utilization. So they tend to use more antibiotics for longer. It's not exactly the same in every analysis, but you can see trends across hospitals, those that are really committed to, for example, antibiotic stewardship and have very short coercerations or things like that, tend to be the same across treatment types and types of appendicitis severity. So perhaps there's hope for reeducating them? Yeah, exactly. I think that's what the last project that we very quickly touched on, which is looking for opportunities to identify best practices at hospitals that are doing it really well, that have low or normal organ space infection rates, but use a lot less antibiotics than everyone else. Thank you. Scott and Savas, a quick question. First congratulations on your piglet model. For those of us who have seen this model, we realize how much work this is. We were wondering, just how much does it cost to have these pace? Obviously it costs a lot near labor, but monetarily. I guess a lot of that comes down to if you're including salary support, if you do not include it. So the first pig, say that I showed, which was the preterm piglet model. We brought three pregnant cows. Each cow had a litter of 13 to 15 piglets, and then we deliver them with lines in, survive them for two weeks, incubators, etc. I'd say for just direct costs, animal costs, things we actually spent, not including anything else, each litter was maybe 50 or 60,000. That's probably a little bit on the low side, but something around there. And the only additional thing I would say is that for our studies, where we did a lot of surgical sections, you have to pay for ORR time in downstairs. It's not as expensive as human ORR time locally, but it does add up. So I think with those, it probably, indirect costs goes up to you, upwards of $100,000 for north of them. Thank you to all three of you. Absolutely fantastic. Docs. I'm going to have to, I do wish we had more time. Each of these could be an hour and then our discussion. Congratulations to all of you. Manesh, you're staying here. Mentors come up, take a quick photo. Well, the veils is walking over to the departmental photo. For those of you on Zoom, I hope you are here physically to be able to make it to the photo in the next 15 minutes. And we will have the rest of you rush over to join. Thank you, Teri, for joining us and we look forward to additional presentations from your colleagues. Thank you. All right, team. I'm going to go. There we go. I'm glad. I know. That's what we are doing. So, what language everyone isο. I'll let you guys. Give me all of this. is right. They are heavy hearts as it works. I haveotes coming up. I'm sorry. I certainly. I'm. Okay. Oh. That was. Like. Yeah. Yeah. Okay. So. Yeah. Yeah. You got to. I'm. Hey, how's it going? That. Thank you. Right. Right. Team. Oh. That. That. That. That. We all know that. It'll. Yeah. Eight. Yeah. Yeah. Yeah. Yeah. Yeah. Yeah. Yeah. Oh, you're making a pause. Yeah. Yeah. Nice. Nice. That. Yeah. Thank you. Hey, Yeah, Yeah. Yeah. Yeah. Yeah. Thank you. Hey, what's up,? you you you you you you you you you you you