Professor Dom D'Agostino: Part 1

Sure. Yeah, my background is undergrad nutrition and biology, and then I studied neuroscience and physiology for my PhD in a pulmonary critical care division department. So I had, in a clinical department, like working, you know, with residents and fellows, actually trained them on research as a PhD student and taught like grand rounds and stuff. And then I did my fellowship, my postdoctoral fellowship, funded by the Office of Navy Research, specifically on oxygen toxicity seizures. So the neuroscience of seizures and preventing, essentially like trying to understand cellular and molecular mechanisms of oxygen toxicity seizures in a warfighter performance program designed to enhance safety, resilience, and performance resilience, I guess you could call it that, in extreme environments. So as you know, special operators are in, you know, extreme environments. It's preventing them from, you know, enhancing their performance and also safety mitigation, especially from a neuroscience perspective.

Yeah, we don't really, well, my research is fundamentally understanding oxygen toxicity seizures. And with a closed-circuit rebreather, which is used by special operations and in some, you know, special operations missions, it is closed-circuit, so there's no bubbles. So the concentration of oxygen you breathe is high. And according to the dive tables, you know, the potential for oxygen toxicity exists with 100% oxygen at just 50 feet of seawater within, within 10 minutes, really. So, so these, these happen, they're rare, but you have situations where a diver needs to go down and plant a mine at, you know, 60, 80, 100 feet, and that's like, you know, that the potential for oxygen toxicity is quite high. And oxygen toxicity is the limitation of hyperbaric oxygen therapy, which we can talk about, and there's 14 different FDA-approved applications. And I was kind of, I like to, we like to do things, we're like on the edge of science kind of, and we kind of like push the limit. So I always had this sort of idea that if we can go from two or three atmospheres, you know, to five atmospheres and actually make that safe, then we could like quickly reverse carbon monoxide poisoning and save a patient. We could quickly reverse gas gangrene. We could quickly mitigate decompression sickness, you know, or as I got into cancer research, we could basically blast tumors that overproduce oxygen free radicals with like five atmospheres of oxygen, you know, if we put them into a high state of ketosis, because that is, that will prevent, and then you have the potential for pulmonary oxygen toxicity. So we work on that, that front too. But yeah, it's, we're funded by Office of Navy Research, which is, and NAVSEA, which is part of the Department of Defense. I also work with Special Operations Command on different things. Now we're working on a TBI thing, and also we've done some work with DARPA in the past too. So.

Yeah, yeah, yeah. We're working with them and on different fronts now. And yeah, they, they have like maybe less funds than Office of Navy Research, and but they're, our work is very in line with sort of many of, and we have very unique equipment in our lab too, which allows us to explore extreme environments.

Yeah, it was a gradual transition and curiosity because I was, you know, trained as a neuroscience and I'm studying oxygen toxicity funded by the Office of Navy Research, and we developed some unique technologies that allow us to look at different cells, tissues, you know, animal models ranging at different models, and also now, now we do human clinical trials with some of the things we developed. But I think it was the observation that I basically, you know, designed something called hyperbaric atomic force microscopy, and then later got another grant to expand that to laser scanning confocal microscopy, which allows us to look at the mitochondria. And in the process of, you know, creating the fluid, gas, and electrical penetrations for this equipment, and then ultimately testing it to show that we could have like sub-nanometer resolution at high pressures of oxygen. I did a lot of different cell types, and one cell type that was very easy to actually study was a human brain cancer cell type. I believe it was taken from a 44-year-old patient. It's a U87 glioblastoma cell line and a few other GBM cell lines. They're just so easy to grow relative to primary, you know, dissociated neurons, which I spent like 10 years like growing them on, plating them on astrocytes, and you know, it's just, it's almost like voodoo sometimes to get, to get really good cells. But cancer cells are easy. I mean, those grow like on top of the benchtop, you know. So we had these cells and we had muscle cells and everything, and we were exposing these cells to graded levels of oxygen. And it was very clear to me that the cancer cells are like super robust and it's like hard to kill them. But under normal conditions, like in the lab, that's, it's an easy model to work with these immortalized cell lines. But I realized, I observed that they were dying fast under a level of oxygen that was relatively non-toxic to primary dissociated cortical neurons and hippocampal neurons and also brainstem neurons. And that was, that piqued my curiosity because

I did not know a whole lot about cancer. I was a little reluctant to go into cancer research, but I just reached out to Moffitt Cancer Center, and I just started digging in, like, why are these cells dying, and could that be something interesting to study, right? Because I did, quite a few PhD students were entering the lab and they wanted to do PhD projects, and one was Angela Poff at the time, or Bennett at the time before she got married, Dr. Angela Poff now, she's my research associate. She wanted to study, you know, these cancer cell lines. So we had the idea of using hyperbaric oxygen with a radioactive conjugated nanoparticle. So the idea was that, you know, because these cells were dying faster, and the Moffitt was telling me that the efficacy of radiation therapy is proportional to the pO2 of the tumor. So the idea then was to bring the radiation to the tumor with a chlorotoxin-conjugated radioactive nanoparticle that would decay in a couple hours. So, not to throw too many big terms out there, but to take something that would bring a pulse of radiation to the tumor in a predetermined time, and that patient could get inside a hyperbaric chamber, and this particular molecule just attaches onto glioblastoma, and it would like pulse the radiation while you're in the hyperbaric, you know, oxygen chamber, and you would have to like radiate through the brain.

So, you know, we put this idea into different grants. It was like reviewers are like, "Wow, this is really cool, but you got to show us some preliminary data," which is always the case, right? So that didn't get funded, but I got more interested in ketones and ketogenic diets because these were preventing seizures. And I was like, "Oh, with brain cancer, well, these patients are often put on like, you know, dexamethasone and anticonvulsant drugs. Well, why don't we just put them in ketosis?" And then there was a 2008 paper that came out of Dr. Thomas Seyfried's lab, and it was like the, in a CT2A model of brain cancer, and he had written a couple other, and then there was a, by the author Nebeling, had a case series report of using in pediatric brain tumors, a ketogenic diet in pediatric patients. So this caught my attention using a ketogenic diet, and I was like, wow, maybe we could, you know, do a project of using a ketogenic diet in a brain tumor model, and then couple that with, since this seems to have some anti-cancer effects, we could couple that with hyperbaric oxygen. And then I had some ideas. We were using lonidamine, 2-deoxyglucose, and a few other drugs that were killing the cancer cells, and that was like little pet projects I would put medical students on. So that was 2008 or 9, I think, and by about 2010, I took on some graduate students and I assigned them to the projects. And I think it was the aha moment was the validation. I reached out to Dr. Seyfried, and he wouldn't give me the brain tumor model, but he would give me the VM-M3 model of metastatic cancer, which I could conceivably put those cells into the brain and make a brain tumor. But he was like, you know, this model of metastatic cancer is unlike anything anyone has ever seen. I think it just made the cover of the International Journal of Cancer. So that was like, okay, this is a pretty legitimate model. And it was, I liked it too because the animal, well, it's good in the way that the animals die kind of quick, like within three weeks.

They get such rapid cancer growth and metastasis that the tumor burden, they usually succumb to the tumor burden and die. So I was like, okay, this is a good way to vet out whether this is going to work. So he sent us, you know, the cells which have a gene in them to make luciferin, luciferase, and you inject luciferin, and the cancer cells bioluminesce based upon, you know, making this luciferase enzyme. So you could, without using like a PET scanner, you can actually image the bioluminescence of the tumor growth, and that was really cool. That was another, and it was like a syngeneic model, like it was an inbred strain. The animals were not immunocompromised, which is a whole other problem with these animal models. It seemed to like overcome, like, you know, we've cured cancer in mice, but there's problems with the models, and this model kind of like that he developed seemed to overcome a lot of these limitations. So we embraced the model, and it worked remarkably well with a ketogenic diet, and then we did ketogenic diet and hyperbaric oxygen, and then we did ketone supplementation with, we did ketogenic diet plus ketone supplementation plus hyperbaric oxygen, and that worked really, really well. And I think we had results unlike anything that he maybe had ever seen. And later, I think he did some work with Dand that rivals that, but it's pretty close. And yeah, and he had done some work, I think, using the model with chemotherapy, maybe methotrexate, maybe cisplatin. But I mean, we went beyond, you know, those results too with the survival. So I was very skeptical, but because of my background, I didn't know that much. In talking with people, they were not super convinced of the cancer as a metabolic disease theory. But the more I read about it, and the more I think it was more experiential, right? So I have a background in sort of biochemistry and cell biology and everything, and what he was presenting made a lot of sense, and there wasn't a whole lot of arguments against it. Like we would have lab meetings and be like, "Okay, critique this theory." And then we would do like rounds. It was kind of hard to critique the way he was presenting it. At the same time, I was also curious why other investigators were not, like, I don't know, embracing it and trying it. So I did not have funding to do this research, but I did get a little bit of startup funds when you transition into a tenure-track position. I had like maybe 50k of startup funds, and we were able to limp along on that. And actually, I think, and then we got like some little grants through industry, and maybe in $100,000, we like published three papers like out of that, and that was pretty game-changing for us in regard to changing my mind that cancer is a metabolic disease. It left many questions, you know, in my mind, like how, you know, we did like the proof of concept, but then it was just like, okay, what about dosage timing? What about other forms of cancer? What about, you know, standard of care? Can this be synergized with standard of care? And I didn't really want to tinker around with radiation and chemotherapy drugs in my lab.

So we made a commitment early on to focus on non-toxic, but well, you know, there's some relatively toxic drugs. So what I guess I would say, not using chemotherapy and radiation if we didn't have to, but we've done with like metformin, we've worked with that, sodium butyrate, 2-DG, lonidamine, and a few other drugs, you know, DCA. I had a student just focused, did a whole PhD project on DCA, dichloroacetate, relatively effective too in the model, but nothing is a, you know, the ketogenic diet is the biggest lever, and then ketone supplementation, even integrated into a standard diet, actually had pretty remarkable effects because by changing metabolic physiology.

Yeah, for me, it was just outcomes-based research. I was like, well, let's see if the animals live longer, and then once we establish that, I'll be interested enough to dive into the mechanisms. So, I mean, over the last 10 years, I've only studied mechanisms. So we've published, you know, beta-hydroxybutyrate suppresses the NLRP3 inflammasome. We studied beta-hydroxybutyrylation, which functions, you know, BHB functions as an epigenetic regulator. We've looked at, you know, GABA levels in response. We do a lot of neuroscience. You know, ketone metabolic therapies remarkably change the neuropharmacology of the brain in a way that can prevent seizures, mitigate consequences from traumatic brain injury. So my wheelhouse has really been mechanisms, but I think we kind of start top down, like we do the research to figure out, you know, what's going to enhance survival, and then we, unlike the NIH model, which is like you find a mechanism and then you kind of, you know, from a very reductionist point of view, kind of work up towards, you know, a therapy. We did the back end of it, and we've done that with ALS research with the Deanna Protocol. Deanna Tadone, who was a patient, Deanna Tadone, still alive today, was only given a few years to live, you know, well over a decade ago. And the Deanna Protocol is a metabolic-based therapy for ALS. And it incorporates, you know, AKG and Ibuprofen at the time and GABA and then some MCT, and that's pretty remarkably effective for ALS. But it was like, you know, the patient was there, and I met with the patient and the family, and then that funding went to the animal model, and now we just, you know, look at mechanisms from there. So.

That one's a hard... Yep.

Yeah. Well, I can tell you that metabolic-based therapies are above and beyond, you know, the standard of care. There's a few websites that actually evaluate these things. The first one, I think, was PatientsLikeMe. So if someone's interested in ALS, just go to the website PatientsLikeMe. Put in the standard of care, which is Riluzole, right? And then look at the feedback on that, and then put in the Deanna Protocol. You get like, you know, the patients, there's about three times higher positive reviews.

You know, proportionally on the Deanna. Nobody knows about it. We've published maybe three papers on it, but, you know, we have not moved that into an RCT, randomized controlled trial. But yeah, I've been in touch with enough patients to know that, you know, these metabolic therapies work in many different domains from seizures, which was the original application. And when I connected with Thomas Seyfried, he said something very interesting to me because I was only focused on seizures at the time, and I read his paper, "Cancer as a Metabolic Disease," or that 2008 study, and I got his email, and he answered the phone, and we started talking. I was quite surprised, and we had a lengthy conversation, and I was just telling him my results, and he made a comment that really, you know, sparked my interest. He was like, oh, and he said it in a very, you know, not an authoritative way, but in a very confident way. He said, yeah, well, ketogenic therapies are more effective for cancer than they are for seizures. So he's like, yeah, you might want to think about, you know, pivoting from seizures to... And that really caught my attention because I was only familiar with the seizure literature. But in 2010, or I started actually in 2005 or 6, just tinkering with these cells and just as like little pet projects, but it wasn't until like 2010 where we actually started doing like animal model research and validating it.

Yeah, he did. Yeah, he was actually chair of the American Epilepsy Society special interest group at the AES meeting, which is more like a for-pharma meeting. And I later became the chair for like three years of the special interest group in ketogenic diets. And then I think my research associate, Dr. Poff, did that too. So yeah, we've always been kind of had that background in seizures, and that's where you learn a lot mechanistically about how ketogenic diets are neuroprotective and have the anti-seizure, and you learn a lot about mechanisms in regard to insulin suppression. So the keto, a lot of people talk about glucose, you know, with, I think of ketogenic diet as insulin suppression therapy, mTOR suppression, IGF-1 suppression, you know, limiting glucose availability, elevating ketones, and then hitting, you know, we look at antioxidant defense mechanisms like the pentose phosphate pathway and some like glutathione, and these tend to go down, and they make the cancer cells selectively vulnerable. So you kind of learn a lot about the mechanisms, and when you learn about cancer metabolism and growth, you realize that you can leverage, you know, this metabolic state, not to cure cancer, I like to be very clear about that, but to create a weaker target for other modalities, right? And I don't think we've actually ever studied the ketogenic diet in and of itself. We've always done the ketogenic diet as like the base therapy to test other modalities. I think that's how it should be. You know, I think looking at ketogenic diet by itself or even DCA by itself is, I don't want to say stupid, but it doesn't make sense. It makes sense more in a systematic way.

Yeah. So with that, I'd like to maybe just pivot a little bit and just talk about like, like prevention, right?

Yeah, I'm just going to mention that like real quick because, you know, we just presented some work to the, you know, at the med school, and there was a big review article that came out in, and it's the journal, basically the flagship, you know, the flagship journal of the American Cancer Association, and it's a, it's over 500 impact factor. So this is like really, it's, I think it's the highest impact factor of any journal, and the title of it is "The Proportion and Number of Cancer Cases and Deaths Attributed to Potentially Modifiable Risk Factors in the United States." And it was pulling from data from 2019, and I think that an important message is that also, you know, in line with psychiatric disorders now in a field of metabolic psychiatry, is that our metabolic health and our metabolic state is a big driver for cancer, and I think that cannot be understated. And we know that obesity is like a huge driver for that. So 50% of all deaths in the United States are linked to modifiable risk factors. So that's something to consider. Metabolic health is tied to nine out of 12 of the modifiable risk factors for cancer. And then, you know, this was not even mentioned, you know, 50 years ago, right? Like nobody talked about metabolic health, and even like the non-diet or metabolic health-related factors, modifiable risk factors that are associated with cancer, even those you could talk about ultraviolet radiation, you know, hepatitis B virus, I think Epstein-Barr virus, CMV, HPV, for example, like these viruses actually cause cancer by modifying the genome to some extent, but these viruses also specifically like laser target the mitochondria. So the mitochondria are easily damaged, and when you accumulate mitochondrial damage, the DNA repair mechanisms in the mitochondria are far, far less robust in repairing that. So we have very robust DNA repair mechanisms at the level of the nucleus, whereas mitochondrial DNA repair is not very good. So you get, you know, progressive inflammation, you get virus-induced mitochondrial dysfunction, and it's more of a protracted kind of thing, right? Like with inflammation and with viral, the virus is attacking that, and the bioenergetic state of the cell, the ATP production, when that gradually declines, the retrograde response, like the nucleus can sense the energetic state of the cell. When it does that, sometimes it dies. But if it survives, it then kicks on oncogenes. And many of these oncogenes are genes that enhance glycolysis. So oxidative phosphorylation will progressively decline with many of these provocative agents that cause cancer, right? That's the oncogenic paradox. You have many different things that can kind of cause the same thing. And I think, you know, the important thing is that it just sort of feeds into the, you know, cancer as a metabolic disease, not only as, you know, as a way to treat it, but it like ideologically it is. So, and that flies in the face of what a lot of people have learned, but I think science is being rewritten, and I think there's a greater appreciation for specifically the mitochondria. Very robust, healthy mitochondria are the ultimate tumor suppressor. So I think that you can kind of put it in that way, and that's why, like, you know, that's why we don't really get the mitochondria in the heart are super robust, right? We look at like cardiac cells, the mitochondria in the muscle is super like robust. In other areas, it could be damaged more easily, right? With progressive, you know, like the liver, for example, with like, you know, hepatitis B virus, or you know, these, you have these organ-specific viruses that affect certain organs and cause chronic inflammation, and that inflammation too is a major driver for mitochondrial dysfunction. So, not to like sidetrack the conversation, but I think, you know, there is no cure for cancer right now. We're working on that, and Thomas Seyfried is sort of spearheading projects that I think will, and I'm really honored to be part of it, spearheading projects that I think will change the standard of care. But I think that prevention is largely the cure when it comes to cancer. And if we appreciate and understand cancer as cancer as a metabolic, genetic, and epigenetic disease, that's how I like to kind of frame it when I'm teaching like the students. But I would actually centralize the metabolic control of epigenetic regulation. So that's something that we study in the lab also. The fidelity of the nuclear genome is intimately linked to the bioenergetic state of the cell. So the capacity of the mitochondria to produce ATP and preserve that, the robust DNA repair mechanisms and the bioenergetic state of the cell will basically by default prevent that cell from the transition from a healthy cell to like a cancer cell. So I think that there's good evidence to support that with some of the research that's essentially the essence of like Thomas Seyfried's book and also what Otto Warburg, you know, presented is that respiratory insufficiency is the kind of the hallmark characteristic of cancer cells, right? You know, and respiratory insufficiency being not respiration but like oxidative phosphorylation, and that's a hallmark characteristic. And, you know, he maybe he was not 100% right about a lot of different things, but he was way ahead of his time.

I mean, aside from the damage that there's there, right? Yeah, they do consume oxygen. I could say that just by, you know, studies doing using the Seahorse Biosciences machine, they consume oxygen, but the oxygen consumption is not coupled to ATP production in the same way a healthy cell would be, right? So, and I think that defines it. And you also have oxygen consumption. Also generating a lot of that oxygen is then goes, which was our first observation, is that, you know, if you push more oxygen through the mitochondria of cancer cells, they produce proportionally more superoxide anion, and we could leverage that in ways to make that superoxide anion more reactive intermediate. So you get, you know, superoxide anion goes to hydrogen peroxide, then hydroxyl radical, and then there's other sort of nasty things like that. And that you can generate from that, and cancer cells will upregulate their antioxidant defenses. And I spent five to seven years studying heme oxygenase. So heme oxygenase one is upregulated in that. So you could potentially use like a heme oxygenase inhibitor. It's probably one of the central defenses that cancer cells use. And they also sequester a lot of iron. So, and this is the ferritin overdrive hypothesis. So cancer cells suck up a lot of iron. A neuro-oncologist or neurosurgeon will use something called 5-aminolevulinic acid or 5-ALA, and they give 5-ALA, and, you know, the patient consumes it, and then they go to do surgery. They do that because 5-ALA is the precursor to heme. So you'll generate a lot of heme, and then the cancer cells will suck up massive amounts of heme relative to the surrounding healthy tissue, and then you could, with a light, you can, it fluoresces, so you can sight specifically pull out the tumor cells. Theoretically, you could, so that actually generates a lot of oxidative stress too. So.

Yeah. And one idea could be, you know, 5-ALA, a heme oxygenase inhibitor, and hyperbaric oxygen therapy. Like this is something like, you know, that I've discussed, I've told oncologists about, and they're like, that makes all the sense in the world. Like, we should do it. We got to write a grant to do, like, this is, these are things that just, I'm just throwing this out there. Hopefully someone will pick up on that, or even like patent it or like do something, just move it into clinical trials because everything that we've done in regard to like little PET experiments basically shows that what I just described would be like largely non-toxic, not toxic at all to healthy cells, and 100% fatal to glioblastoma cells. So if you hit them, and then you could take it another step further and use something like artemisinin. So artemisinin like kills malaria through an oxidative stress mechanism, right? So, you know, hyperbaric oxygen, artemisinin, heme oxygenase inhibitor, you know, in the context of a GKI of one to four, I think, I think by all stretches of the imagination that that will work far greater than anything that we have right now. And what I just described is relatively non-toxic, and a patient could conceivably just get these things online themselves, right? So yeah.

Yeah, that's the idea. You know, get the patient, transition them into with ketone metabolic therapy and use a combination of a modified supplemented ketogenic diet. It's a little bit higher in protein. You supplement that with medium-chain triglycerides, also with a non-alcohol, an alcohol-free exogenous ketone, meaning no butanediol. So pretty much every ketone ester or precursor on the market is 1,3-butanediol based. That could work, but that's, we have some evidence that that could be toxic. So my preference is use a ketone electrolyte salt. Specifically use racemic D,L-beta-hydroxybutyrate with a mix spread across a mix of electrolytes. Audacious Nutrition KetoStart is one, and that we're working kind of in the background on alcohol-free triesters of ketones that do not use 1,3-butanediol that are far potent and far less toxic. So the liver toxicity of 1,3-butanediol is something that a lot of people don't know about, but we're about to publish some papers on that.

Interesting. And I think, you know, so you target, you elevate ketones, lower blood glucose, get a GKI of one to four. Tom's very adamant about one to two, but I think one to four pretty consistent across different models. And glutamine is a little bit more tricky. You could use EGCG, combine that with sodium phenylbutyrate, which is Buphenyl, and that'll help inhibit glutamine a little bit, but also lower circulating glutamine to some extent. And, you know, metformin is something that could also be incorporated. Metformin causes a little bit more superoxide production in the cancer cells. We've published on that. And DCA, is that what it's for? Is that...

Yeah. Yep. Yeah. My student, Dr. Nathan Ward, he has a lab at Moffitt Cancer Center now. He studied DCA and metformin in the VM-M3 cells that Tom, you know, that was his PhD project. So those things relatively non-toxic, readily available. And then some of the bigger guns would be like DCA, and then hyperbaric oxygen is a bit harder to implement. And you mentioned photodynamic therapy. You're probably thinking in the context of the 5-ALA, 5-aminolevulinic acid. And yeah, I was not even thinking in that direction, but that could be, you could photodynamically activate too. I'm thinking about the patient when the head is not open, you know, to do that. So the idea that with the 5-ALA, the artemisinin, and the hyperbaric oxygen, you could do that independent of photodynamic activation. But you bring up a good point with photodynamic activation, you could probably go, wow, you could get like a huge burst.

Yeah, it's a shorter wavelength. It's in my research proposal. I should...

know that, but I don't have that at my fingertips. And I'm hesitant to quote because I'm sure...

Yeah, I thought about methylene blue too. Because I have some of that in the lab and I was thinking that could potentially protect the cancer cells. I had a lot of questions about light and I've talked with, what's his name? The light guy. Smart nerve surgeon guy.

He's a genius.