Hi, everyone. Thank you for joining us. I'm Lily Dara from the University of Southern California. It's my pleasure and honor to introduce Dr. Laura James for the ASLD state-of-the-art High Zimmerman hepatotoxicity lecture of 2023. Laura is very well known to those of us in the Dilly world for her seminal work in acetaminophen hepatotoxicity. She has pioneering work specifically in the detection of protein adduct measurement. Laura is a native of South Carolina and obtained her medical degree from the University of South Carolina. She completed a residency in pediatrics at the University of Arkansas Medical Sciences and a fellowship in pediatric emergency medicine at the University of Alabama-Birmingham and a second fellowship in pediatric clinical pharmacology and toxicology at the University of Arkansas for Medical Sciences. She has been focused on studying acetaminophen and acetaminophen toxicity detection for the last three decades. Her research program in acetaminophen toxicity has been funded by the NIDDK since 1999. In 2006, she co-founded ATD or Acetaminophen Therapeutic Diagnostics LLC to develop a novel laboratory test, the Acetastat, for the rapid detection of acetaminophen toxicity. ATD LLC is funded by the Small Business and Technology Transfer Program of the NIDDK. Dr. James is currently a professor of pediatrics and the Associate Vice Chancellor for Clinical and Translational Research at the University of Arkansas for Medical Sciences. She also directs the Translational Research Institute where the CTSA Award resides and is one of the one of the 60 centers funded across the U.S. Please join me in welcoming her. Thank you so much, Lily. It's such a privilege to be here today, and I'm really excited and honored to be selected to give this lecture as a memory and tribute toward Dr. Heye Zimmerman. The title of my talk this morning is Detection of Acetaminophen Liver Injury in 2023. So Lily really kind of shared my background. I have been in Arkansas for almost 30 years and been fortunate that I was able to grow and develop as a faculty member there, beginning in pediatrics, but then working and aligning very quickly with those in pharmacology who were also interested in acetaminophen liver injury. So I think this is already disclosed that I'm part owner of a small business known as ATD. We have been funded by the NIDDK through their small business grant mechanism, and my collaborators, Dr. Dean Roberts and Jack Hinson and I have worked together over this time period, and we do have a patent that has been granted by the U.S. Patent Office for a methods patent for measuring acetaminophen protein adducts. So just a brief outline of what I want to cover today, just a brief introduction about how I got interested in this topic, then we'll move into the role of oxidative metabolism in the generation of adducts and in toxicity, and then I'll review some of our work with HPLC EC assay, and then close with work on the immunoassay, which we are calling Acetastat, talking about how we developed it, and then currently reviewing our current progress. So I am a pediatric pharmacologist that as a junior faculty member in the mid-90s got somewhat taken aback by the lack of diagnostic tools for acetaminophen liver injury, and this interest really came out of the clinic. We had about three patients in over one winter that developed fulminant hepatic injury. Of those three, two resulted in death, and I remember being struck by the irony of this, that we had a medication that was very commonly used, very widely accessible, and we knew how to make the diagnosis in the early stages of toxicity, but in the later stages we were kind of left to clinical judgment only, and so that really kind of started my interest in acetaminophen toxicity, and the second point of this slide is to make, really to lay out the importance of interdisciplinary team science and translational research, which really has kind of characterized my career over the last three decades, even really before I knew what I was doing and before I knew I was engaged in translational research, and then I also want to give a huge nod to NIH-supported clinical research networks, which have been so important in my career, as well as a small business grant funding from the NIH. So this slide, I'm sure, is known to everyone in the audience that works in the daily field. These are data published by the Acute Liver Failure Study Group in 2007, and of course we see the very, very large contribution of acetaminophen to acute liver injury in the U.S., and although this slide is somewhat dated, I don't think that the contribution of acetaminophen or any of these other etiologies have significantly changed. So I know at this meeting and at multiple meetings we've heard a lot about newer mechanisms that are important in acetaminophen liver injury, but the point of this slide is not to stress these factors, oxidative stress or mitochondrial injury. The point is to recognize that most authorities working in this area really recognize the role of oxidative metabolism as the initiating step in acetaminophen liver injury. This slide is also a dated slide, but it's still very relevant for this talk and relevant for understanding of acetaminophen liver injury, showing the important role of P450 oxidation, whereas the formation of N-acetylparabenzoquinoneamine, or NAPQI, which binds, it was a highly reactive metabolite, which binds to cysteine groups on protein to initiate the toxicity. And of course we know that hepatic glutathione is very important in detoxifying NAPQI, but with large doses of acetaminophen, glutathione is depleted and there is a relatively increased formation of NAPQI. Data reporting relationship between formation of acetaminophen protein adducts, toxicity, dose, and time were very well explored in the mid-1980s, and this is just a snippet of that data, a paper published by Neil Pumford showing that after toxic dosing of acetaminophen in the mouse model, liver adducts very quickly rose and then several hours later there was the appearance of serum adducts. I don't show ALT data here on this slide, but if this were present you would see ALT values increase slightly before the formation of acetaminophen protein adducts in the serum. So this is in contrast as we move to the clinic, this is the standard approach for making a decision about whether or not a patient has ingested enough acetaminophen to warrant treatment. This is the REMAC nomogram first published by Dr. Barry REMAC in 1976. Most of you are probably familiar with this slide. It's much more familiar to emergency department physicians who really live by this decision-making tool to make a decision about whether someone has ingested enough acetaminophen to require treatment with an anecdote inositolcysteine. And we know that this works very well for the patient that comes in early, gives a clear history, takes an acute ingestion, and is overall relatively healthy. However, there are numerous limitations to the REMAC nomogram and this unfortunately is more common than I think than people realize. People that come in whose time of ingestion is unknown or they're unaware of the poisoning, the staggered ingestion, the ingestion that's a chronic overdose, and then patients who use other medications or already on other medications or ingest co-medications, and then patients that have pre-existing liver disease. So these are the known limitations and I'll just you can't probably can't see this x-axis very well but this is the 24-hour line and out here we go to the 32-hour line. So this really is not that helpful for patients that come in later to the to the hospital which is where the hepatologists get involved. So in 2002 our laboratory published a high-performance liquid chromatography electrochemical assay for the quantitation of protein adducts. The principles of this assay are that a blood sample is obtained, typically separated as serum, is treated with gel filtration to remove low molecular weight molecules including acetaminophen conjugants and acetaminophen as the parent drug. The sample is treated with proteolysis precipitated filtered and then the remaining sample contains amino acids including the acetaminophen cysteine. The sample is then injected on the HPLC machine where on the basis of retention time, electrochemical characteristics, and quantification of the add or the peak relative to an authentic standard curve of acetaminophen cysteine we were able to report out a concentration of acetaminophen cysteine or acetaminophen protein adducts. This next slide shows a relative example of chromatography from a patient with acetaminophen cysteine adducts and this is a retention time of about seven and a half minutes. If acetaminophen was still in the sample it would elute out at a little bit after eight minutes but you can see that there's a nice distinct peak here showing the presence of acetaminophen cysteine. So the first time that we really took this assay and married it up with the clinical setting was through a collaboration with the acute liver failure study group. I'm going to list several article titles and show you the authors. I don't have really time to go through but this has very much been a collaborative effort with the acute liver failure study group. The first question was can we detect adducts in patients with known acetaminophen liver injury group A and then group B was acute liver failure cases of known other etiology. Group C were patients with acute acetaminophen overdose who presented to the ER and received treatment with NAC early. So clearly we were able to detect adducts in those with known acute liver failure where for the other two groups they were either negligible or not not present. The next step was to ask about well what about that group of patients that have acute liver failure of indeterminate etiology and you can see with groups D and E here there was a subset within that group that also had a similar profile for adducts, similar median and range of values showing that the first glimpse that this might have a diagnostic role in identifying patients who previously were the etiology was unclear. So we really used this assay to learn more about adducts in different clinical settings. This is data from that first study the Davern et al paper where we found 19 percent of adults with acute liver failure of indeterminate etiology had adducts that could be detected. Similar findings in children slightly lower percent and then the third bullet was a pharmacokinetic study showing that the elimination half-life of adducts is very long. This is 1.72 days not hours and if you know about the pharmacokinetics of acetaminophen this is much longer than the known PK for acetaminophen and then we did two studies where we tried to make sensitivity and specificity comparisons with patients with known acetaminophen overdose and acute liver failure and found that an adduct level of 1.1 was highly sensitive and specific in these acetaminophen patients with an ALT of greater than a thousand and these values were 95 and 97 percent. A subsequent study that was a larger study we were able to kind of round this down to 1.0 and again looked at patients with acute liver failure of unknown etiology. We also wanted to know what is adduct performance or what adduct levels look like in patients with therapeutic or low-dose exposure, and I'm just gonna show you two examples here. First study was a simulated study of acetaminophen overdose with a lower dose of acetaminophen where there were multiple samples obtained which allowed for pharmacokinetic analysis, and the peak level of adducts in this kind of low-dose exposure study was 0.1, so we were a full order of magnitude below the toxicity cut point that I showed in the earlier slide, and then a more recent study conducted with Mitch McGill, we looked in patients with compensated cirrhosis and compared those to healthy control patients. Both groups of patients received two grams of acetaminophen daily for four days, and we measured pharmacokinetics on day five. Patients came in at day five, and we did 24 hours of PK sampling in these patients, and you can see that, again, the range of magnitude of adducts is from 0.4 to 0.8 for the healthy. A little bit higher in the cirrhotic population, but closer to 0.1, and I see John Dranoff is in the audience, and this was also, he was also a collaborator on this study. So the next question is can we take this to the clinic and can we turn this into a diagnostic, and these are the grants that helped us to do that. Again, these were STTR grants through NIDDK. The very first study was can we just make a homemade dipstick, can we do this in the lab? This is the work of Dean Roberts, and just to orient you to the slide, the lateral flow assay, you have an application pad on the left side, a test band, a control band, and a wicking pad. So the serum will flow from left to right. The control band indicates assay integrity. The test band, because this is a competitive immunoassay, at this test band line, there is acetaminophen cysteine antibody, and when the antibody binds to cysteine groups in the sample, there is disappearance of a line. So it's a little bit opposite of how a urine pregnancy test works. We're actually looking for the disappearance of a line, and you can see with the HPLC-EC measurements, as they get higher, the test band disappears. So this was really our first phase one study. We showed that we could make a lateral flow assay, and then this is our second study where we worked with BioassayWorks to create a device, a cassette, that could be tested in a clinical study. At this point in time, we were using a polyclonal antibody. In this version of Acetastat, things are kind of flipped from what I showed you in the earlier slide, but again, sample application pad, control band, and test band, very similar in the construction to what I showed you on the prior slide. This study was published several years ago, again, another collaboration with the Acute Liver Failure Study Group, in which we shared Acetastat devices with these sites and also showed that with a reader, we could measure adducts in patients outside of our lab. So there were 62 patients with ALF, 19 helpfully volunteers. Patients contributed a single blood sample. The sample was split and tested with Acetastat and a reader, and then the rest of the sample was measured by HPLC-EC. This is just to show how the data broke out. 19 controls, 33 with acetaminophen, 29 without acetaminophen, and we are looking in these acetaminophen cases, we're looking specifically for a lower test band amplitude. So you can see the difference here between the acetaminophen, the non-acetaminophen ALF cases, and then the healthy controls. And then the same data shown with the HPLC-EC assay. So this slide puts all of that data together. Little bit hard to read, probably the acetaminophen liver failure and injury cases are in the right upper quadrant. The non-acetaminophen cases and the controls are in the left lower quadrant. And we see we have a few patients that didn't really play by the rules, but by and large, there was a very strong agreement between the acetastat and the HPLC-EC assay. So this study really demonstrated as proof of concept that we can do this at other centers. Again, this was a polyclonal antibody with a strong agreement of 95.1% of cases. So there were several things that we really needed to do to move this forward to commercialization, and I'm gonna go through these very quickly, that this has really been the substance of our work for the last three or four years. BioassayWorks was not a GMP, GLP lab. We needed to transfer the technology to a lab that really made lateral flow diagnostics for FDA, to enter the FDA approval route. We found a great company in DCN Diagnostics located in Carlsbad, California. We needed to develop a new antibody. Our polyclonal antibody was made in a rabbit, but we needed to have an antibody where we knew exactly what the antibody sequence was and where we had a reagent that was a defined reagent that we could use over and over again. We also needed to have an authentic standard. Cayman Chemical was able to validate acetaminophen cysteine and declare it as a certified reference material. So this is also needed for the FDA approval route. And then we needed to meet with the FDA. In the very first meeting that we had with them, they said, well, you've got to validate your HPLC-EC. If you're gonna call that your reference or your gold standard, you need to validate that. So we completed that work in 2019, and we needed money. So we received two other grants also from NIDDK to continue this work. I want to take one step back just to kind of review a lateral flow assay. And it ended up that while it seems like a simple thing to build a lateral flow assay, there are actually multiple considerations that go into the development of these assays. There are three key reagents. First, we had to have an antibody. We needed a great test band reagent, and then we needed many, many samples with known concentrations of acetaminophen protein adducts. And again, I'm showing you a typical acetostat figure here. And then also, as we look at this and getting to making these for potential commercialization, there are multiple variables in both the physical components and the liquid components of the assay. So for the physical components, there's the sample pad, conjugation pad, nitrocellulose membrane, a wicking pad, and there are probably 12 to 14 variables or different options for each of these. And so as we went through this work with DCN, multiple iterations and options for all of these were tested to find something that really gave consistent performance and what we called kind of the best mode for acetostat. Liquid components included things like buffers, volume, pH, salt, and sugar. And again, multiple different components of these were tested to determine the best mode acetostat. This is a very busy slide, but I'm going to walk you through it just so you see an example of the types of experiments that we've been conducting with DCN in this development stage. So this is our test line and our conjugate conditions. You can see that there's different percentages of sugars, different concentrations, the sample treatment. We had to test the sample volume, look at flow rates, et cetera. The rest of this slide is really showing either the HPLC-EC concentrations and or the density of the control band and then the test band or test line. This is an axon image, which is just a visual image, and then the individual that was in the lab testing these samples also gave it a visual score. And you can see as you go down, it may not project very well, but we really want this disappearance of the line to occur at around one. You may be able to see, some of you may be able to say that here's one here where it looks like there's a high level of adducts, but still a faint, faint mark at a test line. This is another example of a type of test that we did. Again, the blue is the test line intensity. The red dotted line is the axon cutoff. And this is really the point at which the visual, at which the eye, the human eye, can no longer see a line at the test band. And we, of course, we wanted this to be at a value of 1.0, and you can see that we're getting pretty close here. There's one sample that's kind of bumping up against that red line, but this is just an illustration of the types of studies that are done as we develop the best mode for acetastat. So currently, well, let me back up and say that about eight months ago, we got to a point where we realized that all of our banked frozen samples, we had exhausted those, and so we took a pause and opened up a new IRB protocol just to obtain fresh samples. We had questions about sample validity for samples that had been in the freezer for many years. And so we took a pause, have collected new samples, and have about 140 samples that we have now shared with DCN, and we're kind of resuming that development stage. We're very close to what we think will be the final version of acetastat. We also have contracted with other academic centers because there's going to be the need over the next year to have many fresh samples, and this is going to be important as we scale up production of acetastat, go into usability testing, and then complete in vitro trials. We need to go back to the FDA for a final review of the clinical trial design. We really can't do that until we have completed these in vitro studies, and then we're hoping that by the end of, close to the end of 2024, we'll be ready to launch what we hope will be a definitive clinical study that we can take to the FDA for approval. So there have been many groups, many individuals, many NIH-funded networks, and others that have participated in this work, and I want to thank them with this slide, and thank you for your attention, and again, thank you for the invitation to be with you here today. Thank you.

Dr. Laura James

pediatric pharmacologist

ASLD state-of-the-art High Zimmerman hepatotoxicity lecture

acetaminophen liver injury detection

Acetastat

acetaminophen toxicity detection test