#272 ‒ Rapamycin: potential longevity benefits, surge in popularity, unanswered questions, and more | David Sabatini, M.D., Ph.D. and Matt Kaeberlein, Ph.D.

Hey everyone. Welcome to the Drive Podcast. I'm your host Peter Attia. This podcast, my website and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness and we've established a great team of analysts to make this happen. It is extremely important important to me to provide all of this content without relying on paid ads to do this. Our work is made entirely possible by our members and in return we offer exclusive member only content and benefits above and beyond what is available for free. If you want to take your knowledge of this space to the next level, it's our goal to ensure members get back much more than the price of a subscription. If you want to learn more about the benefits of our premium membership, head over to peterattiamd.com subscribe my guests this week are David Sabatini and Matt Kaeberlin. Matt has been a former guest on three occasions, episode 222 and 175 and all the way back to episode 10 while David was on the podcast way back in episode number nine. Not only are they two of the original guests on the podcast from our 12 part pilot experiment in the summer of 2018, they are also two of the most knowledgeable people on this planet. On the topic of conversation today Rapamycin and MTOR in my conversation with Matt and David, we cover the discovery of rapamycin. We look at how mtor, which sits at the epicenter of our existence, works and does its job. We talk about the pathways of rapamycin that are believed to drive its impact and improvement on lifespan. We discuss the initial studies that showed rapamycin may be zero protective and also what studies have come out since or are currently in progress which provide more information and clarity around this very important question. Finally, we discuss what is known and unknown about the potential frequency and dosing of rapamycin in humans. I'm very excited to release this podcast because I don't think there is a question I get asked more about from my patients than this topic. Understandably, because my patients know that I take rapamycin and have been doing so for about five years. It's understandable that they want to understand if it's something they should be doing too. And I think you'll see from this discussion why I have reservations about just blindly putting people on rapamycin. In other words, why is our practice not a rapamycin mill? I hope that this podcast is helpful not just for my patients, but everybody listening, including Everyone who is interested, of course, in this question. So without further delay, please enjoy my conversation with David Sabatini and Matt Kaber. Well, guys, we're going to try something a little different today, which is we're going to try to have a three way discussion, which is something we would easily be doing if we were sitting over a meal, but always makes for a slightly more challenging podcast. That said, given our familiarity with each other and your familiarity with topic, I am 100% confident this is going to be an amazing episode. It's also an episode that is long overdue. So you guys are both in the camp of, I believe, first dozen or so podcasts that were released on the drive a little over five years ago. Matt, you and I have spoken a number of times since David, you and I, at least on the podcast, have not. Obviously in person all the time. So with all that said, many people are going to be new to this topic. They will have heard a lot about it. They may have even read a chapter about it, my book, which you guys were both very gracious to help me fact check and edit. But here we are. We're going to pretend that someone coming into this discussion doesn't really know anything about rapamycin, doesn't really know what this MTOR thing is. I hope that by the end of this discussion we will have provided people with arguably the most comprehensive, quasi concise explanation of all you need to know about said topics. With that said, I would like to ask each of you to do something I don't often ask my guests to do, which is toot your own horns a little bit about what it is that allows me to say, you are each among the two most knowledgeable people on this topic. Let's start with you, David. You've worked on this molecule, rapamycin, your entire scientific career, going back to your PhD, and here we are, 30 plus years later, you're still the leading authority on it. Can you tell us a little bit about that?

Sure, Peter, thank you for having us. Nice to see you, Matt. So no, indeed. I've worked on rapamycin my whole life. When I was a student with Saul Snyder or Johns Hopkins, I became fascinated with this molecule and frankly, I needed a research topic. And so I tried to figure out how it works. And that led to the purification of protein that we now call mtor. Michael hall had identified a yeast version of this that Matt was one of the early workers on this, called Tor1. And since that time we've done a lot of biochemistry try to figure out what this protein does, and at the end of the day, what we conclude in a kind of big picture point of view is that this is the protein that links the availability of nutrients in our environment to whether we're in a catabolic or an anabolic state. Anabolism, growth, catabolism, breakdown of material. And I think that accounts for why MTOR has so many different roles. Because if you. Throughout our sort of evolutionary history, there's almost nothing in our physiology that shouldn't be controlled by the availability of nutrients. It's such a central thing in our lives. We tend to forget that now because, of course, we're in a overeating stage. And since that time, what we've done is figured out a lot of the pieces of this pathway, including what we call two complexes, protein complexes, MTORC1 and MTORC2. And really the work that I'm the most satisfied with is how it senses nutrients and the nutrient sensors themselves, which are the actual proteins that bind the small molecules that tell mtorc1 in particular that it takes nutrients. And so I'm excited to be here and to delve into some of the implications of this work.

Awesome. Thanks, David. Matt, people who are listeners of this podcast are going to be maybe a bit more familiar with you because in addition to that very first podcast we did circa 2018, you've been back a number of times and we've talked about MTOR and rapamycin, but we've also talked about protein nutrition and things like that. But maybe for folks who are hearing you for the first time today, can you give a similar bio of what it is that allows me to also refer to you as one of the world's absolute leading authorities on this topic?

Sure.

First of all, thank you for having me back. I'm glad you aren't sick of me yet. It's fantastic to be here with David. And I wish I could say that I was smart like David was and recognized immediately how important MTOR would be and rapamycin. But I actually started working on MTOR in yeast kind of by accident. So we were really interested in understanding what are the genetics that control longevity. And so we did an unbiased search for new genes that would affect lifespan and happened to find mtor. And when we made that discovery, I immediately went and looked up everything I could learn about MTOR and found out there's this drug, rapamycin, that's an inhibitor of mtor. And then we found that we could also increased lifespan with rapamycin at this Point, we were working in yeast, but then it became clear to me, because of the work of others, that this pathway and this drug appeared to affect the biological aging process, not only in yeast, but also across the animal kingdom. And we now know even in mammals like mice, and potentially in larger mammals like dogs and people. So I think with that knowledge, I got very interested in trying to understand what the mechanisms are for how rapamycin was affecting the biological aging process. We've studied this in yeast and worms and fruit flies and mice, a little bit in pet dogs, which we may talk about. And I think through all of that, the one thing that has kept me excited about rapamycin as a potential longevity therapeutic is that it always works. And I would say, without question, it is the most robust and reproducible drug, at least from preclinical studies that we know about today, for impacting not only longevity, but to the extent that we can measure various metrics of health span in complex animals. Rapamycin also seems to positively impact pretty much every aspect of health span that we measure. So I've continued to study it for that reason, and I think probably what I'm maybe best known for these days is pushing forward a veterinary clinical trial of rapamycin to really start to answer the question of all the things we've learned about rapamycin in the context of aging and longevity in the laboratory, how much of that will translate into the real world? And so we are actually carrying out a veterinary clinical trial of rapamycin in pet dogs right now. We've got some preliminary data, but it's too early to be able to say, you know, with any level of confidence that rapamycin is going to positively impact the aging process in dogs. But I think we've already learned a lot about safety and maybe some hints about efficacy. So that's pretty exciting. And that's something that I am quite passionate about continuing to push forward and see where we end up.

And I guess I should, before I make my next comment, disclose that I and a number of my patients are funders of a study that we will undoubtedly talk about. So we should just, I guess, declare that as a conflict, if people want to consider that a conflict. But regardless, I think what's really great about having you guys together and tell me if you agree with this assessment, is on the continuum of understanding rapamycin and mtor. David, you're closer to what we would call the bench side of things. And in many ways, Matt, I consider you kind of closer to the bedside. So people have Heard this term bench to bedside. That is translational research. And obviously, the bedside in this case is not just the bedside of humans, where we aren't quite yet, but really the bedside of more complex mammals. So would you guys kind of agree with that assessment that your skill sets and your knowledge base and your research are very complimentary through that continuum of bed to bedside?

I would. I would add, though, that Matt takes the work in a very serious scientific way. Right. And so I think in a field where it's very easy to get caught up in boosterism and claims that you see online all the time that are extreme, I think Matt has been very careful. And part of what's contributed to the interest in rapamycin, frankly, is that Matt has been careful about this. And so he's taken a very scientific approach. And as I've told many people who know me well, I pretty much put Matt in one of the most respected categories of aging researchers for that reason.

I agree with that completely.

Guys are embarrassing me. Come on. Now, Peter, let me just give a little bit of a twist on what you said. I mean, I agree with what you said. Conceptually. I would also say, I think even though much of my research on MTOR and rapamycin has been what people would typically consider preclinical or basic research, it is different. I agree. I think the approach that David has taken throughout his career is quite complementary in many ways to the approach that I've taken in that David has really, as you already said, been the. The pioneer and the leader in really understanding detailed mechanistic aspects of the whole MTOR signaling network. And I don't know how much we're going to get into that, but I think it's useful for people to appreciate that this is an extremely complex network of biological interactions. And there's no question that David and his lab and people who have come from his lab have really played the leading role at fleshing out, from a very detailed biochemical and mechanistic perspective, how that network is working. And that has, I think, in many ways, laid the foundation for people like me and many others who have then taken that knowledge and tried to start to move it into maybe more applied contexts and clinical applications.

That's a beautiful way to describe it. And I also want to echo something that David just said, and again, not to embarrass you, Matt, but I do think that the field owes a lot of its credibility to the way you have approached it, with scientific rigor being the highest priority, as opposed to the commercialization not being front and center. And I do think that there are a lot of other molecules that maybe we don't have to get into today where there is some interesting science behind it. But it seems to have almost been corrupted by a commercialization route. And the corruption of that has meant a we may never know if these things work or don't work. But more than anything else, they're very difficult to take seriously. And I think everybody should be very grateful for the way the field has gone. So before we dive into it. And I know this is a story that's been told before on this podcast and I probably even write about it in the chapter of my book. I do think that the discovery of rapamycin is the place to begin this because there's a very unique phenomenon here, which is the drug was discovered before the target and the target is named after the drug in response to that. So, David, you and I got to visit this very special place where the bacteria that ultimately produced this drug was discovered. We certainly have plans to go back. It's on our list of things to do in the next few years and we shall. Do you want to give folks the story of how this molecule rapamycin came to be?

Sure, sure. And I hope we'll go back soon and I hope Matt will come as well.

He certainly will.

Anyone who cares about rapamycin for the.

I did try to get this recording done on Rapa Nui, so I just want to put that out there.

We'll be doing another one there for sure.

As you know, Peter, there were and are attempts by pharmaceutical companies to collect soil samples and other biological containing samples throughout the world. And Wyeth Ayers did come into possession of a soil sample from Easter island, otherwise known as Rapa Nui in the South Pacific, at one point claimed to be the most remote island in the world. I think it's actually technically not, but very far from anything. And in that soil sample, actually in Canada, people eventually isolated bacteria from it, bacteria called Streptomyces hydropicus. And from that bacteria, rapamycin was eventually isolated and in deference to Rapa Nui, was named rapamycin. Now, ironically, it turns out when people have looked for rapamycin and other bacteria throughout the world, in fact even the same bacteria, it actually has been found in many other places. But it did come originally from rapamycin and like was done at the time these molecules, these bacterial products, you really would call it an antibiotic. It did come from. Bacteria were tested, you know, in many different assays. And I Actually think, and Matt may correct me, I think some of the earlier assays were actually immunological assays, even before some of the antifungal assays. And that eventually led many decades after to pursuing it as an immunosuppressant. But in the meantime it was also found to have antifungal agent activity. And that's where some of the genetics, arapamycin and some of the targets were first identified because of the ease of genetics. So this is a story that began, I think the original soil samples may have even been in the 60s.

Yeah, I think it was 66 or 67 soil samples. And then Suren, I don't think really got around to digging into it until 71 or 72.

Exactly. And then he championed it. In fact, one of my most valued possessions when I started working on rapamycin, we didn't have much. And Saul Snyder, my advisor, wrote Saren and asked for some. He sent us many grams, which I later calculated had a street value of many hundreds of thousands of dollars, if one could sell it like that. And a really nice note, wishing me luck. And the entire bibliography of Rapamyce at that time, which consisted of his papers and a couple of abstracts, is a little thin book at the time. And he is the one who championed it. The clinical path took way too long and I think that even impacted some of its utility because the patents expired, I think, before you really sort of capture some of the value of it. So we're talking about something now that's in the 50 year range plus and I think a question that we could ask ourselves, I think we will. Is rapamycin as good as it gets? There obviously are derivatives of rapamycin, but even in this pathway, which as Matt says, exceedingly complicated, are there other targets that we should be pursuing that may actually have equal or better impacts on the aging process?

Can I just add one thought, something David said there we may again also touch on which is the clinical path not only took too long, but I think you can make an argument that the clinical path has actually maybe negatively impacted the development of rapamycin and other mtor inhibitors for other uses. Because it was developed clinically as an organ transplant immunosuppressant and that's how it was first approved. It was used in a dosing protocol and a patient context where there are lots of side effects. And I think we are still learning what the side effect profile actually looks like for rapamycin at lower doses in patients who are not immunocompromised. And haven't had an organ transplant. So I do wonder whether the history of rapamycin and the rapidity at which it will be eventually tested for other endpoints in clinical trials where it may have benefits has been negatively impacted and slowed down because of the reputation that the drug got as a dangerous drug based on the way it was developed clinically. So I think that's an important piece of the puzzle here to think about.

Just to give some numbers to it. The first paper that Suren Seagal put out there describing the chemical composition of rapamycin, if I'm not mistaken, was about 1971. 1972. The FDA approval for rapamycin in humans was 1999. Just to give you a sense of what you're both talking about here in terms of an enormous gap of time between when you sort of make a chemical discovery, file an IND and work all your way through. Second point I'd make is, as a former surgical resident, I was in my surgical residency taking care of transplant patients when rapamycin was in full use. Now, again, it's interesting, David, the whole reason you got involved in rapamycin was because of FK506, which was a cousin of rapamycin, that, if I recall, the whole reason your lab was using rapa was as a control that didn't have sort of the calcineric properties of FK506. But that's an interesting footnote. But we were giving rapamycin out constantly. And to your point, Matt, it was a drug that was typically given 2 to 3 milligrams a day every single day. But with three other drugs, you are also getting Prednisone, Cellcept, MMF. You are getting very, very toxic drugs because you needed to completely shut down the cellular immune system of a patient who had just received a foreign organ. And I think that speaks to this point, which is for the better part of a decade, 1999-2009, the only experience the scientific world has with this is in that context. Yeah, you're going to see a lot of side effects. But how do you know they're from rapamycin? And how do you know that they would be the same elsewhere? So what happened in 2009 that kind of changed this? And, David, I'm most interested, I think, in hearing this from you, because by this point, you've already established your own laboratory. You're working on rapamycin, you're working on mtor, probably more so than anything else, and trying to understand the nutrient sensing pathways around it. But how aware Were you of the itp, the Interventions Testing Program, in the buildup to that first study in 2009?

I was not very aware of it, I have to say. But I do have to say that once we started making that connection of rapamycin to nutrients, which many groups did, if you actually look at the history of it, and it was already appreciated for many, many decades before, that things like caloric restriction had an impact on lifespan. So the idea that rapamycin could have an impact on lifespan was one we actually had thought of. And we actually. This just tells you how science works. We actually tried dosing C. Elegans worms with rapamycin, naively not realizing that their cuticle would not allow rapamycin the way that we were giving it to have an impact. We had found no impact. And then there were genetics that came out in worms and Matt's work and a lot of other people really pioneered the aging space, not us at all. But I remember when that paper came out, I think it was a Nature paper that came out reporting rapamycin as one of the bigger hits in the IDP study. And I think what happened there, I think Matt said this before, it connected his work in yeast and work in other organisms with a mammal. Now, we just take that for granted, right? Because as Matt said, it does impact all those different animals and single cell organisms. But the idea that we had a molecule that spanned from a yeast to a mouse was dramatic. That was like a huge, huge impact. Again, we take it for granted.

Now I'd like you to say more on that, both of you. I do think that the evolutionary gap from yeast to flies, worms, mammals is a billion years. Are there any other molecules that have done what you just said, David?

I don't know if there are, but certainly dietary restriction, yes, in one form or another. And that did link all those organisms. And as far as I know, it was all done before rapamycin, before the discovery of tor. So there was this universal intervention. I think even in bacteria, people have shown impacts on replicative lifespan. So that, I think, was considered this universal connector. And that's why when the nutrient connection came out, I think we and others started thinking along the lines of rapamycin as an emetic and potentially having this impact. So I don't know, Peter, where there are specific molecules that do that.

I'm not aware of any. I mean, yeah, I'm not asking the question rhetorically, but I agree with you that outside of caloric restriction, which by the way, doesn't universally extend life, there are Models and certainly times at which that can be administered when it is not a life extending strategy. But yes, I mean, rapamycin in that sense stands alone. Unless, Matt, you can think of a counterexample that I'm missing, I'm going to.

Try to respectfully tell you guys that you're wrong. So here's what I would say. Rapamycin for a small molecule is probably the only pharmacological intervention that has been reproducibly shown to robustly increase lifespan and health span across that broad evolutionary spectrum. There are other things out there like alpha ketoglutarate, where there are reports in yeast and worms and flies and mice of lifespan extension. It just hasn't been tested or reproduced as much. But on the genetic side, and this is where I wanted to just add a little bit of additional context to what David was saying, which is that with rapamycin it's not only the drug, but we also have genetic inhibition of MTOR in each of those model systems that recapitulates the longevity and healthspan benefits. So it's a rock solid, airtight case for MTOR and longevity. But also on the genetic side, this is a study that we did with Brian Kennedy and Daniel Promisloe. This is probably 2007 where we asked the question, if we looked at all of the genes at that time that were known to affect lifespan in yeast and all of the genes that were known to affect lifespan in worms, and we simply looked at orthologs, meaning the same gene in each organism, how often is genetic control of longevity shared? And it turns out it's pretty often. So there is a relatively high degree of evolutionary conservation at the level of genetic control of longevity across a broad evolutionary distance. And that's really been the whole thesis of my career, right, is trying to understand those evolutionarily shared mechanisms of longevity. So I just think it's important for me, I guess, to say that, because there's a lot of confusion now in the field. There have been a lot of new people come into the longevity field who for whatever reason aren't familiar with a lot of this history. And so they ask questions like, well, how do we know that you can use worms to understand anything about aging in a mammal? And I'm like, because we already know that the genetics of longevity are conserved, not everything's going to be conserved, but it has been statistically shown that there is a conservation of the biology of aging. And that's kind of fundamentally important to how we think about studying the biology of aging. In the laboratory and then potentially translating those discoveries into the real world. So again, sorry for the tangent, but I do think this is an important, more fundamental biology of aging point that's useful to just re emphasize because a lot of people have lost track of that.

I completely agree, Matt. And when I just saw Peter in Austin and he took me on a rocking Trek in 104 degree heat, we exactly talked about this topic. And my point was that biochemical cell biological processes that are conserved amongst all these organisms are going to be the ones that are going to impact aging. And in fact, I don't. I tend to dismiss those processes which are less concerned as potentially causing impacting the aging process. So I 100% agree to you that whatever is the fundamental issue that happens in cells that leads to aging is going to be conserved. And therefore the regulators of that process or the impactors of that process will be conserved.

I want to come back to this point. If we had been able to record that ruck session, if you could eliminate all the huffing and puffing, it would have been a great podcast in and of itself. And we're going to come back and talk about some of those things. This is actually a great step off to make a point that what we are talking about here is the broad term of zero protection. And I always kind of differentiate this when I'm talking to my patients. I say there are certain strategies that we take to extend your lifespan and improve your health span that are very disease specific. So for example, the attenuation of apo lipoprotein B is undoubtedly going to lengthen your life if implemented for a long enough period of time, and by extension, I would argue, improve the quality of your life. But it's doing so through two disease processes. It's doing it through a reduction of atherosclerotic cardiovascular disease and cerebrovascular disease, but also through all lines of dementia. But it's not attacking a fundamental pillar of aging. It's a very disease specific hack, for lack of a better word. And by the way, it certainly wouldn't be applied to organisms beyond ourselves. Very few organisms have apob I. E. Very few organisms succumb to ascvd. That doesn't mean that we shouldn't look at disease specific tools to modulate lifespan and health span. But what we're talking about here is so much more fundamental. I will not put either of you guys on the spot and ask you if you can recite the nine hallmarks of aging. Matt and I tried that on our Last podcast and got.

I can get seven. I think we could do this, like name that tune.

Right.

How many hallmarks of aging can you.

Right, right. But there are nine of these hallmarks of aging.

There's actually 12 now. Right. With the new and improved version.

Oh, there is. My God, I'm so dated. Okay, so let's now dive into MTOR a little bit.

Can we go back to that mouse study?

Go back to the itp? Yeah, sure.

Yeah. Because there's another part of that study. So just for a little bit of context, I'm not sure this ever got said explicitly, but this was a study from the NIA Interventions Testing Program published in 2009. It was the first study to show that rapamycin treatment in a mouse could extend lifespan. And that was important. But I think the other, maybe more important part of that study that often doesn't get always talked about is that this was the first time that any intervention. You could argue a little bit about caloric restriction. That's kind of a tangent. But I would say it's the first time that any intervention was convincingly shown to extend lifespan when treatment was started in middle age. So about the mouse equivalent of a 60, 65 year old person biologically. And that again, as David said about rapamycin, we kind of take it for granted today that that's possible. But in 2009, I don't think anybody expected that experiment to work. It was actually an accident that they ended up doing the experiment that way. And it had to do with the fact that they couldn't formulate the rapamycin in the mouse child in a way that was stable until the mice were already about 12 months old. So treatment started. I think in that case, it was 20 months of age when they finally started the treatment. So it was a happy accident. But in my view, I've said this before, I think this is one of the most important studies in the field in the past 20 years, maybe 50 years. For that reason that it opened up what we now consider to be routine, which is that you can actually have an impact on longevity and some metrics of health span when you start treatment in middle age. And as we have started as a field to think about translational application, that becomes hugely important because suddenly we're talking about treating middle aged dogs or middle aged people as opposed to trying to treat puppies and teenagers. And that's just much more pragmatic and practical from the perspective of actually being.

Able to implement, especially when you consider what David said at the outset, which Is MTOR is the master regulator of how nutrients trickle into the system. Are you going to be in an anabolic state or are you going to be in a catabolic state? Well, Matt, you'll be pleased to know we just got a puppy recently. So we've got this adorable little three month old puppy. And I don't think it would make sense to necessarily inhibit MTOR in an animal that is purely about anabolism. Right now it's trying to grow and it would be suboptimal if we had a therapy that we believed could only work if administered early in life. And yes, your telling of that story is remarkable and I think also speaks to the serendipity that often lies in scientific discovery. It's often an accident or something going wrong that leads to that. And I've talked about this, I think with Rich Miller, they were contemplating sacking the whole study because they couldn't get the rapamycin formulated.

It is a fascinating question though, why the starting point of delivery of rapamycin does have an impact on the life extension and health span extension and what the biological basis of that is. Something that at least I don't have a great conceptualization of that. And I'm sure Matt has thought about this much more. But it's interesting to think about how one designs experiments to try to ask that question.

Is it safe to say we don't know when the ideal time to implement would be?

I think it depends a little bit on what you mean by ideal. Right. So this now gets into risk, reward and side effects versus benefits in mice. We absolutely don't know in terms of lifespan. If we take that as the primary metric that we're interested in, we don't know when is optimal to initiate treatment or what dosing protocol is optimal. So there still has not been a full, what I would call even dose response profile of rapamycin across a single intervention time point, initiating time point. The answer is no. And honestly I think we probably never will. Simply because the cost of doing those experiments and all the permutations that you could come up with for time that you initiate and different doses to test, I just don't think anybody would ever fund that study. We're getting off on a tangent, but it's probably worth just mentioning that going back to the starting in middle age, this is where I actually have some real concerns with the way we fund biomedical research in general. If somebody went to the NIH before this study had been completed and said we want to start an experiment with rapamycin starting at 20 months of age in mice, that grant never would have gotten funded because people would say that'll never work. And so this is where I think again, it was very fortunate in this case that it happened the way that it did. But I would argue, as a research enterprise should develop an appetite for higher risk, higher reward projects. And I don't think anybody's going to disagree with that. But I think this is a nice case in point of a. An important discovery that changed a field that would not have been made if not for just the fortuitous circumstances that happened.

Yeah, I think that's completely fair. I would challenge you on one thing though, Matt. I can't think of a better type of research to fund for relatively low dollars than the types of questions that you're asking. In other words, I agree that there are a lot of permutations and I agree that we're talking about tens of millions of dollars. But when you consider what's at stake, I. E. What we could learn, I guess for the listener it's worth explaining something. We're going to come back and talk about this and we're going to talk about intermittent dosing. But these ITP studies are dosing rapamycin every day. It's mixed into the chow. So the mice are constantly nibbling on a low dose of rapamycin. And what we're going to go on to talk about as we start to extrapolate into, for example, companion dogs and ultimately humans, is a dosing regimen that looks completely different. Well, for starters, I sure as heck would like to see what that looks like in the mice of the itp. I'd also like to see some of these different permutations around the different, not just doses, but starting points. And again, if it cost 10 million to do that study, I gotta tell you, I think we could raise that money. It didn't take too long to raise half that money to do the dog aging project. So I think there would be a real appetite to do that kind of work because the implications are enormous. The NIH might not fund it, which is probably what you meant.

That's right. I think also, and we may get into this as well, there are a bunch of those kinds of fundamental questions that I would argue are relatively low hanging fruit. And then we would have to think about prioritizing. I think we're going to talk a little bit about Rapalogs or other classes of MTOR inhibitors. The other classes of MTOR inhibitors. There was just recently, the first study that I know of that tested an ATP competitive MTOR inhibitor in mice. It's intriguing, I would say early data, but we really have no clue, as far as I can tell, how other classes of MTOR inhibitors would perform relative to rapamycin. That's another super important question that again, for frustrating reasons, has been very hard to get those kinds of studies funded. And I can just tell you from my own experience, I have put in grants to study dose response of rapamycin, different intervals of rapamycin testing, and other classes of MTOR inhibitors, and they have been uniformly rejected because by and large, NIH study sections just aren't interested in funding those kinds of studies. They're not considered, at least in my view, they're not considered mechanistic enough. So I agree completely with you, But I think those kinds of studies will not be funded in the current structure for research funding, even though they're super important.

Yeah. So, David, let's now start to talk about the how. I think it's worth doing this in a little bit of detail. And I know that the next few minutes might be among the most technically, perhaps challenging for a layperson to understand. But I think it is important to have some understanding of the biochemistry of what this molecule does and what this protein complex looks like and what the cascade of events are that move on. And I think it's also important to understand how nutrients work. So we're going to talk a lot about amino acids and probably in particular leucine. So in any order that you feel it makes sense to walk us through that, David, explain how this molecule, mtor, which sits at the epicenter of our existence as living entities on this planet, how does it do its job?

One thing for the listeners to understand is that rapamycin is quite unique in another aspect that we haven't talked about, but also was very exciting at the time. Right? Rapamycin, unlike most drugs, most drugs go and find their protein target and do something, usually inhibit that target. Rapamycin gets in the cell, binds to a little protein, fkbp. What it does to fkbp, frankly, doesn't seem to matter at all, but instead hijacks that protein and now takes it and makes it bind to mtor. It basically uses it as this thing that it draws next to mtor. And that moving of FKBP to MTOR is actually critical for how rapamycin acts, as people like Stuart Schreiber have pioneered. It's really a molecular glue that connects MTOR and fkbp, and that interaction is absolutely critical. So how Does MTOR work? When we first and others found mtor, it was this big protein. It looked like a kinase that is, it's a protein that puts phosphates onto other proteins. But yet what it did, what its targets were, were completely unclear. And I think as we were talking in the pre session Matt pointed out it's incredibly complicated. It probably acts on hundreds of other proteins in general. What are those other proteins? They're either proteins that make the cell build things, this anabolism side, or break it down. And on the breaking down side, as you and I, Peter have discussed, I'm sure Matt agree, it's autophagy, right? The self eating and destruction of parts of the cell, sometimes aged parts of the cell, sometimes parts that are damaged for other reasons. That seems to be absolutely critical on the catabolic side and the way MTOR works. For a long time we had mtor, we couldn't really get it to phosphorylate anything in a test tube. It just didn't work. It seemed like a terrible kinase. That is its enzymatic activity was so puny we even thought maybe it's not really a kinase. It really was like a moribund protein. And the critical breakthrough was the idea at some point that MTOR must work by being bound to other proteins. Now again, this seems obvious. Everyone talks about the TOR complex, but at the time it wasn't. And the reason was that of course we and others had looked, we'd said, okay, isolate mtor. Does it have friends? And the answer was no, it has no friends. What we came to realize, and this goes back to serendipity, it turns out the detergents, when you have a mammalian cell, it's surrounded by a lipid, fatty membrane, you have to break that to do biochemistry. It turns out the detergent we were using, which was the most commonly used detergent to break cells for simply bad luck reasons, broke apart the MTOR complexes. You could never predict this and why it does it we don't know. And when we moved to other detergents, used things to stabilize it. We then found these TOR complexes and the first breakthrough for us was the discovery of a protein that, that got this name Raptor. At the time people didn't like this name and now is a well studied protein. And as Matt alluded, there's actually genetics on Raptor that connect it to lifespan and the aging process. And so that defined what we now call TORQUE1 or MTORC1. Another protein that we named Richter defines what we call MTORC2. I'm sure we'll talk about MTORC2 at the time. And so we started building out that complex. And now when you had that thing in a test tube, it did stuff. It could show serious activity that you could measure. It could do serious phosphorylation, the known substrates like S6 kinase, that before we couldn't phosphorylate S6 kinase to save our life inside a test tube. Now all of a sudden you really could. So it really opened up the door. And then that connected MTORC1 to all the other things that in sort of a biological lingo we call upstream. All the proteins that communicate to mtor bring signals to it, are upstream of it. The things MTOR acts on are downstream of it. And we've actually done very little downstream. I would say we really focused on the upstream. I would say the next big conceptual breakthrough for us came when we looked inside of cells and saw that MTOR was in a particular place. And this is a organelle called the lysosome. The lysosome is sort of the recycling center. This is where a cell takes things and breaks them down and releases nutrients. And so it turned out that MTOR lived at this very interesting interface where the cell produces its own nutrients by breaking down things, and also where the nutrients are coming in from the outside at that intersection. And we went on then to find lots of the pieces that allow that nutrient sensing. And I'm sure we'll get into amino acids and other nutrients afterwards.

David, if I can interrupt for a sec, Approximately how many MTOR complexes exist in a typical cell? And let's talk about maybe what the typical cells are. What's the distribution of MTOR concentration across different cells in the body, things like that?

Yeah.

In terms of numbers, we're talking certainly thousands of complexes in existence. It's not an amazingly rare protein. It's not incredibly abundant at all. You know, it's probably in the hundred to a thousand fold less than some of the most abundant proteins in the cell. The proteins are much, much less abundant than that. And it's distributed actually quite evenly between MTORC 1 and 2, at least in the cells that we have looked in culture. When you look across tissues in a mouse or a rat, it's actually pretty even across tissues as well. And so to some extent that puts it in the what sometimes pejorative years called a housekeeping protein.

How uninteresting, right?

Exactly. Some of the most important proteins in the cell. What we have found now, and I think others would agree is that regulation of MTOR levels itself doesn't happen that much. It does, but it's not the critical regulatory input, it's all the upstream stuff. And the regulation of that, that really is where the pathway gets fine tuned in different cells to different inputs and where I think we have to start thinking about also for new modalities to target mtor.

We'll park this idea of tissue specificity down the line. But if I'm hearing you correctly, even though I don't know that people have sampled the CNS of humans, based on what we know from mice and rodents, of rats and things like that, we have reason to believe that you would have comparable MTOR concentrations within CNS tissue, peripheral tissue, probably everything. I'm guessing virtually everything except a red blood cell or maybe even a red blood cell. Do we know if it's in the RBC as well?

There actually is some in RBCs, which has been very confounding to us because RBCs don't have things like lysosomes in them. There's even some in platelets or mitochondria. I've actually always wanted to go and look in RBCs for this reason. As far as we can tell, every cell has some MTOR and MTORC one. And I would argue, and I'm not sure if I'm 100% correct in this, I would argue that Almost every cell, MTORC1, is a very critical protein for the health of that cell. And Matt alluded to a study, I guess, where people have used now catalytic inhibitors. And we need to distinguish that what rapamycin does. People call it an allosteric inhibitor. It binds to mtor, but it doesn't bind in the heart of mtor. If the heart is where it does its phosphorylation reaction, that's sort of like the central node of it. It doesn't bind there. It actually binds close. And what it does, it prevents certain substrates from getting to that kinase domain. It kind of sterically blocks them from getting there. So it doesn't fully inhibit all the activities of even MTORC1.

So let's give people an analogy, David. So, for example, in this case, if the amino acid is like a baseball, that's supposed to bind inside the glove. Rapamycin, by blocking that, doesn't sit itself right in the heart of the glove. It maybe binds outside the glove and closes the glove. It changes the shape of the glove so that the intended target doesn't. Is that a good analogy?

It is. Now the Thing that binds in the glove here is ATP, which is the phosphate donor, and then the substrate, let's say, ESSEX kinase. You're exactly right. ATP can get in there no problem. It's small, it can easily get there. But what happens is, basically, it's almost like the entrance to a cave. And now you've put a boulder in the entrance of that cave, but you haven't fully blocked that entrance. So, simplistically speaking, some small things get in there, some smaller substrates can get in there, but some bigger ones can't. And there's also, of course, as you alluded to, shape changes and stuff. But the simplest way to think about it, it's a steric block of some things but not others.

Perhaps also worth just re mentioning that this is the mtorc1 cave, which is again, different from the other classes of inhibitors which are going to affect MTOR in Both MTOR Complex 1 and MTOR Complex 2.

And, Matt, you said that there's been a study now on lifespan, or at least aging, writ large with catalytic inhibitors. This is actually something I've always wanted to do because they're extraordinarily toxic molecules when dosed at higher levels. So I'll be curious. I've not seen this, but you're right. The catalytic inhibitors basically annihilate the activity of MtORC1 and MtURK2. If used at the right dose, rapamycin partially inhibits MtORC1 and over time can also partially inhibit MtORC2. So they're very dramatically different.

Can you say a bit more about that latter point? It's a very subtle point, but it's going to come up again when we talk about the difference between continual dosing and intermittent dosing. What is it about the kinetics of rapamycin's inhibition of MTOR complex one that will eventually, but not immediately lead to the inhibition of MTOR complex 2?

Before I'll say that MTORC1, its canonical substrate is Essex kinase. So every biologist looks at ESSEX kinase phosphorylation as an indicator of MTORC1 activity. The canonical substrate for MTORP2 is a protein called AKT. Everyone looks at AKT phosphorylation as a canonical output. And so I had this postdoc, one of the more colorful people, I had a guy from Kazakhstan, actually, Das Sarbasov, who had discovered Richter and the AKT phosphorylation. One day he comes to my office. He's like, David, rapamycin inhibits Mturk2. And I was like doss, that is impossible because we had tried to show that this FKPP rapamycin would bind to mtorc 2 and it wouldn't bind. It would bind fine to mtorc 1, but it wouldn't bind to mtorc 2. He comes, he shows me data, he's like, look, if I use rapamycin for a long period of time, I inhibit AKT and I also break apart MTORC2. And I didn't believe him, frankly, at all.

What year was this, David?

This would have been early 2000s, and somewhere in that range, I'd have to look back maybe 2003, 2004, in that range. I think we published the paper maybe in 2005. But this is one of those cases which I'm sure Matt has experienced many times too, where the trainee really is driving the story and convinces you of what turns out to be a pretty important discovery. But I didn't believe. And so the reason was, why did this happen? Right? Because you could take mtorc2, you could put fkbp rapamycin in it, and mtorc2 would phosphoryl akt, no problem. It didn't care. Totally fine. You do the same experiment with MTorp1 and S6 kinase, and now you could really inhibit S6 kinase phosphorylation. So what we came to realize, though, and to some extent it's obvious, is that of course MTOR2 is not born as MTORC2, it's born as MTOR and Richter, and they have to find each other. But what happens is that FKBP rapamycin can bind to mtor, what we called naked mtor, it can bind to it, and when it's bound, it turns out that Richter can't bind. So you can't make MTOR 2. And so what happens then, Peter, is that when you incubate a cell and a mouse over prolonged periods of time of rapamycin, all your mtors acquire an FKBP rapamycin, and therefore you can't form a Richter complex. And so the way that we're preventing MtorC2 formation and therefore MtorC2 inhibition, is completely different than how it impacts MtorC1. It's basically preventing the biogenesis, the formation of MtorC2. So you need these two proteins, Mtor and Richter, to come together. Basically, FKP rapamycin is preventing that interaction. And the way people are getting around this, which I think we're going to discuss, is by understanding that at a better biophysical Level, which we now do have that understanding.

So, Matt, given what David just said, does it surprise you that the ITP study and many of the studies that have looked at constitutive dosing of rapamycin have still managed to find a longevity benefit?

No, it doesn't surprise me. But I think the reason it doesn't surprise me is in part, I think we need to again recognize that this network is extremely complicated. So the model that David laid out, I think is kind of our best guess for how this is working. And I agree, everything he said is correct from a biochemical perspective. What the impact is on the overall network of transient rapamycin treatment at a given dose versus chronic rapamycin treatment at the same dose or a different dose is much harder to really understand in a detailed way. So part of the reason why I'm not surprised is because we kind of already knew all the longevity outcomes before we understood this biochemical mechanism. And so now we're trying to work backwards and say, how do we explain the fact that rapamycin can increase lifespan in a bunch of health span metrics, given that the way it was dosed in the mice should have also impaired MTOR Complex 2? And built into that is the assumption that the reason rapamycin is extending lifespan and affecting health span metrics is purely because of the MTORC1 inhibition. And I would say that piece we don't completely know. The best evidence for the idea that the benefits of rapamycin come from mtorc1inhibition is the genetic data, which we've sort of alluded to in yeast and worms and flies and mice, where you can mutate proteins or genes that code for proteins in MTOR complex 1 and see lifespan and healthspan benefits, but that's incomplete. So I guess it's all to say that I think, and this is dissatisfying to me and probably everybody else out there, but I think it's true that we still don't fully understand the mechanisms by which MTOR inhibition and rapamycin can impact the biology of aging. And therefore we're working with incomplete models. And I'm not convinced at this point that the idea that all of the benefits are due to MTORC1 inhibition and all of the side effects are due to MTORC2 inhibition. I'm not sure how accurate that model is. It's a model that still needs to be studied.

So I completely agree with Matt. I think that last statement is 100% true. I think we almost have no evidence to make that decision one way or another. But I think the reason if MTORC2, its inhibition is toxic, which we have published papers arguing it is, the reason that I think it's actually quite tolerated is because, you know, in general, the amounts of rapamycin used in the longevity studies are relatively modest. They probably still are somewhat intermittent, even though a mouse is eating them. Right? Because of course, it doesn't eat all the time. But unlike what we were doing experimentally, where we were dosing rapamycin very high, keeping it above a certain level, and certainly in tissue culture, it's 24 7. And you can imagine that once an MTOR finds a Richter, it's immune to rapamycin now. So as soon as one of those guys interact, you're going to have an MTORC2, and you need very little MTORC2 to keep AKT happy. We found that early on, you only need probably 10 to 15%, at least in cells and culture to keep AKT happy. So there's going to be escapers. As soon as rapamycin goes below a certain amount, there'll be escapers, and you'll make an mtorc, too. I do think we have to ask how relevant that activity is to the potentially beneficial effects of rapamycin. And a lot of the drive to find rapamycins that don't do that comes from my work. Right. And so to some extent, I am saying, hey, look, is that oversold? I think that is a potential argument to make. And Matt alluded to, I would almost argue there's no perfect experiment to answer this question because MTOR is shared. You almost can't answer this. Almost a philosophical issue.

One thing I want to add, David kind of said this, but I think it's really important for people to appreciate because sometimes we get into the routine of talking about MTOR and MTORC1 and MTORC2 as if they were on off switches, but they're not. They're kind of like, you can think of them as knobs. And so what David said about you don't need a lot of MTORC2 activity to survive. And the same thing is probably true for mtorc1, but rapamycin is turning down mtorc1 immediately a lot. And that's going to depend on the dose of rapamycin that we give and then over time, turning down the mtorq2 knot, but it's not going to zero. And so, again, it's important that people appreciate that it's not an on off, and that's part of what makes it really hard to do the definitive experiment that David was saying we can't really do given the tools we have, because it's so complicated. And the tools we've got are not clean in that context, even though they're very biochemically clean.

And there's tremendous feedbacks, Matt, that fight all of that. The system always is trying to get to homeostasis.

So, David, talk a little bit about discoveries that were made in your lab about what the amino acids were doing to mtor, because those actually are things that were learned much later than the initial discoveries you made around the interaction between rapamycin and mtor. So what do we know in particular about branched chain amino acids or leucine in particular?

So this also has a little bit interesting backstory. So when I first identified MTOR in Saul Snyder's lab, I talked to my dad, who is a cell biologist, and he said, david, you have to localize MTOR within the cell. To be honest with you, I kind of dismissed that in maybe a little bit in an arrogant way, because I was like, look, I'm a molecular biologist, biochemist. Cell biology is kind of an old thing, but we did actually make an antibody to mtor. And, you know, at the time, we used to make them ourselves and rabbits, we had some. We purified, and I added it to cells, and it gave this very interesting punctate pattern inside the cell. And I remember walking around the cell biology department at Johns Hopkins Medical School asking people, what is this? And I didn't get any definitive answer. Then the rabbit died, the antibody was lost, and literally for about that would have been in 93 or something, literally until probably 10, 15 years later, we did not revisit this question. It was Tim Peterson in my lab who did. And when he did do this, and he did it in a definitive way, the answer was lysosomes, as I mentioned.

Sorry. Just to make sure we understand, David, you're saying when you did the original experiments with the antibodies and they lit up, and you're walking around showing them to people saying, what would light up in this pattern? It wasn't clear what the answer was. In other words, it wasn't clear where MTOR was.

They saw dots inside the cell. Now it was clear that there was a little vesicles. And I think probably, you know, if I had sat down, this was like, literally walking around the hallway. So maybe if I had sat down with more experts and really showed them more experiments, we would have gotten a more definitive answer. But that didn't work. And then, you know, you go on. And again, literally, the rabbit died. The antibody disappeared. I would say, no good antibody to do this experiment for the next 15 years. And this guy Tim got one, and he showed again. We saw the same punctate pattern that I had seen as a student 15 years earlier. But he then went on to figure out what it was. And they were these things called lysosomes, Again, these sort of recycling centers. These are compartments in the cell that have a membrane. Things get in them. And there's about 60 enzymes in that compartment that can basically annihilate anything, break it down into single components, like, for example, proteins. Amino acids come out, polymers of sugars, individual sugars come out. And that was fine. But the critical experiment and the one that really changed everything for us is then Tim did a simple experiment. He said, well, let me remove amino acids and look where MTOR is. And it turned out it wasn't on lysosomes anymore. It went off the lysosome. Then he added amino acids, and he had even little movies. Within minutes, it went back to the lysosomes. And so what that told us is that nutrients communicated to mtor, and one of the things they did was move MTOR to the surface of the lysosome. And then we went on and we found the docking station. So it turns out you can think of MTOR as like this big ship, and there's this docking, like a pier. And when it gets there, it sits on top of these proteins that hold it there. And it turns out that those proteins are the ones that nutrients talk to. And there's an entire set of proteins about. I think I counted there's about 20 proteins involved in making that communication to drive MTOR to the surface of the lysosome. And we could go into the details of this, but it's probably a little bit too much. But there's multiple large protein complexes that do that communication. And. And what I think that indicates, and I've said this in talks, could have been simple, right? There could have been one protein, binds an amino acid, talks to mtor, but it's not. There's a lot of protein real estate used to do this, which tells you the cell cares about this. So the question becomes, which amino acids? And I have to say that really, that was broken open not by us, but by Joe Averick. He had a paper in JBC where he looked at amino acid regulation of mtor. This was before the lysosomes. He was looking at the Activity of using essex kinase. And he basically found a couple amino acids that mattered. He found leucine very common essential branch chain amino acid, an important component of whey protein, for example, that people take arginine, a very basic amino acid, technically not essential. Lots of nitrogen in that amino acid. And those were the two big ones that he found out. Since then we have found others. And to us, the holy grail was how is leucine detected? That was the thing we wanted to know literally for decades. And the reason was, is that there's a lot of literature in mice, in humans, in big animals used in farms that leucine does cool stuff like boost satiety, feeling of having fed, boost muscle mass. And eventually we found it. We found the receptor for leucine. It's a protein called cestrin. And for me, you know, you have, in your scientific career, I think you only have a couple of moments where you're kind of moved because you see something and you've been hunting it for a long time and you see it, and for us was the crystal structure of leucine bound in cestrin where you're like, okay, this is how nature does it. So from eating a steak to now detecting the leucine in that steak, there it is, it's nestled in there. And then you could sort of imagine how it goes on. To talk to mtor was this Bobby.

That led this work.

So Rachel Wolfson and Lynn Chantranoopong, they had discovered cestrin as the sensor for leucine and they could show that genetically, biochemically. And then Bobby Saxton working with us and Thomas Schwartz, he then did the crystal structure of leucine bound. And what was beautiful about that structure was it immediately said it's gotta be leucine, which we and others had shown already, right? You could try isoleucine, but it didn't work. And so you could see it nestled in there and you could see all the parts of cestrin that said it's gotta be leucine. The sobering part was it's a small little pocket. Leucine is a small molecule, very small molecule. And so it's not clear how you can mimic. The immediate idea was, hey, can we mimic the anabolic effects of leucine without taking leucine? Can we make something better than leucine? And we've managed to make things slightly better, but nothing dramatically better. And the structure tells you why, because it basically is made to fit leucine and nothing else.

How long does leucine Stay in that pocket?

We don't know, Peter, but it's an interesting question because the pocket, there's a little pocket and leucine binds, and then there's a lid that falls on top, so it literally closes it. The evidence suggests that getting leucine in is easy, getting leucine out is not easy, and that there actually may be an active way of getting leucine out. That lid has some very interesting sequences in it that suggest that it might be phosphorylated to sort of pop it open. So we don't have an answer to that question, but I think you hit upon something that suggests that it's not the leucine is popping in and out. It pops in, but probably getting out requires an active step.

So, Matt, how do we reconcile two things that seem a little bit at odds here? On the one hand, we've just established that MTOR is the most important sensor we have, not just for nutrients, but perhaps more importantly, the most critical nutrients of them all, which are amino acids. We also understand that sarcopenia is an enormous risk to both lifespan and health. Spanish sarcopenia, meaning low muscle mass. So we understand the relationship between amino acids and muscle mass. We understand anabolic resistance in an aging population. So all of these things say amino acids are good, MTOR activation, that is anabolic activation is good. And yet we've just made a very compelling case for why blocking that extends lifespan. How would you start to reconcile what seems conflicting?

Obviously, it's going to be extremely complicated. I think I'd start by going back to a point that I made five minutes ago or so, which is that these are not on off switches. So you really need to think about this in the context of what is the optimal level of MTOR complex one activity for whatever it is that the cell, the tissue, the organ the organism needs to do to function or stay alive. So certainly we know that you need MTOR activation to build new muscle. And so the idea was that rapamycin treatment inhibiting mtor, turning down mtor, should lead to faster muscle loss. That was the prediction that was made so that rapamycin should induce sarcopenia. If you were to treat animals with rapamycin as they were getting older, that was the prediction that was made. The reality turns out to be the opposite, that it seems to be the case certainly in rats, probably in mice, we don't have data yet in people, frustratingly, but certainly in rodents, that you can treat them with rapamycin throughout adulthood and actually preserve muscle mass into old age. So the explanation for that, I would say, is still a little bit unclear. Almost certainly it's going to be at least partly dose. If you were to give them too much rapamycin, you probably would in fact accelerate sarcopenia. But at the doses that have been used to increase lifespan, it seems like you can actually preserve muscle mass during aging. That's a different question, though, than I think, which is one that a lot of people ask, which is if you were to take rapamycin, would it prevent your ability to build new muscle mass? And it might if you're a bodybuilder. I don't think we have any data in humans on people who are just doing resistance training in the context of just wanting to maintain muscle mass or build a little bit of muscle mass as they're getting older. I just don't think we have that data. I don't think we have the data in rodents to really answer that question either. In the context of the doses that extend lifespan, would that impair the ability of those animals to build muscle mass if they were put on some sort of a resistance training regimen? I don't know that anybody has done.

That experiment yet, which is a shame, because it's been done with metformin. There's no reason we shouldn't know that question, right?

Sort of. So metformin you're talking about in human studies.

Yeah, in the humans, yeah.

So people have looked a little bit. A little bit. Although, again, I would say even there the data is not definitive yet. But you're right, there have been some studies where people have looked at the effects of metformin on exercise, both resistance training and cardiovascular training. Yeah. I'd say the data is unclear, although there is some reason to think that metformin might impair what people think of as the positive responses to exercise. Complete tangent, but I agree with you. The fact that that hasn't been done for rapamycin in humans is a shame, and it should be, and hopefully will get done sometime in the near future. I wish I could tell you why that's the case. I'm just sort of telling you what the observ are. My intuition is that part of this comes down to the effects of rapamycin on chronic inflammation, which we also know increases with aging and can impair synthesis of new muscle as well as preservation of existing muscle. And so I think you've got some competing interests here in that rapamycin inhibition of mtor complex 1 by rapamycin, it might actually somewhat impair synthesis of new muscle Even at the doses that seem to promote longevity in rodents, but it might actually preserve muscle because it's having this more broad anti inflammatory effect. And so this is why I think it's hard to get to a specific detailed, mechanistic answer to your question because people haven't really started to disentangle those things. The last thing I'll mention is that I'm a little bit wary of extrapolating too far from the rodent studies to humans in the context of sarcopenia in particular. And in particular I'm talking about mouse studies to humans. Mice are not, at least the common, commonly used inbred mouse strains are not particularly prone to sarcopenia with age. There are some rat models that are better. And so I worry a little bit about the use of mouse models in particular to try to say this is or is not going to have an impact on sarcopenia in humans. And I'm not talking so much about rapamycin in this context, but I'm talking more about the studies of protein restriction and branched chain amino acid restriction, which in mice seem to have some positive effects on longevity. But because mice, they don't develop sarcopenia to the same extent or in the same way that people do, I would worry a bit about extrapolating from that to say that it's going to have those same beneficial effects in people where sarcopenia seems to be much more important for quality of life, probably life expectancy, but certainly quality of life in older adults. So I just want to make that caveat. We need to be a little bit careful about extrapolating from mouse studies to humans in the context of muscle preservation, muscle function and sarcopenia.

I think that's actually really important. It's certainly one of my gripes with people who tend to over index on protein restriction in animal studies, which is A the model itself, B the environment in which the model exists. If you're living in a sterile environment where there aren't curbs to step off and places to fall and injure yourself, I mean, one only need look, look at the mortality data for people over the age of 75, even over the age of 65, if they fall, it's an enormous cause of not just death, but morbidity, total destruction of quality of life. I want to ask you both a question, or you can both chime in and whoever has a stronger point of view on this, maybe I'll start with you, David. Do we know from the laboratory in mice, for example, what the tissue specificity is of rapamycin do we have a sense that we are getting uniform MTOR blockade, or do we get the sense that, you know, no, it's disproportionately happening in the liver or it's disproportionately happening in the adipose tissue. I mean, because this would factor into it. In a dream world, you might construct a version of a rapalog that also has some tissue specificity in addition to what everybody's talking about, which is complex one specificity. So, David, anything you can add on that?

Matt answered your question perfectly well, and I think it shows you the complexity of the issue. It's not only MTORC 1 versus MTORC 2, it's which cell type is it muscle fiber? Is it inflammatory cells, immune cells? At what dose is it? Which process, autophagy, is it protein synthesis? So these are very complicated questions. Now onto your question, Peter. Certainly if you dose it high enough, in our experience, you will inhibit mtorc1 in all tissues that we looked at. It takes a little bit of time. If you're talking about classic rapamycin, to get in the brain, typically need to do a little bit of a loading dose, but you'll get it into the brain out. There's been some discrepancy. Some people say immediately in our hands, it usually took a couple of doses to get in the brain.

Was that a couple of doses without interruption?

Yeah. Typically where we did not let a trough level to get too low, this would have been probably in a mouse, maybe every eight hours or something, maybe every 12 hours. So it's pretty aggressive type of dosing.

So your view based on those data, if you were extrapolating, is that if you were taking rapamycin weekly, you're probably.

Not getting CNS penetration, probably with classic rapamycin. And there was some, you know, in terms of the pharma world, people that wanted to treat tuberous sclerosis, where you get these tubers in the brain, they did not think rapamycin was adequate for that because of CNS penetration. But again, very talented people have argued differently than that. But our experience certainly was that the brain seemed more resistant. In fact, sometimes you would stain the brain, you'd see almost like a peripheral inhibition, like it kind of had permeated a little bit from blood vessels in the dura and stuff. But I think the more relevant question, Peter, is at these lower doses that people take, potentially for healthspan lifespan studies, in the ITP studies, what are the tissues that are most affected? Matt may know, but I don't know. And my feeling is that it's not going to be so equal in those situations. Those are very low doses compared to what we would give to rapamycin. My bet is that there's much more variation and that might actually be very interesting to know. A critical study that has been done in worms with other modulators of aging, which as far as I know has not been done in a mammal for the MTOR pathway is if genetically we inhibit MTOR in the muscle, in the liver, in the brain, which one has the most prolonged lifespan. Healthspan impact Matt I don't think that.

Study'S been done, not systematically. There's a little bit of data on hypomorphic MTOR alleles and gosh, I wish I could remember the outcomes of these. Veronica Galvin's done some stuff for dementia, brain aging. I don't know about lifespan. I think Torin Finkel may have done something, but in general it has not been done outside of if it's been done. There was like an adipose specific knockdown knockout, maybe liver specific, but certainly not systematically looking across different tissues.

This does get at this question is like, okay, there's lifespan, but then there's also the health of all the different tissues. And my bet would be that you actually want to impact all tissue. Now I used to go to aging meetings and I would always challenge when at the speaker dinner I would say, tell me a biological system that does not age. Give me one where you don't see the impact on aging from the biochemical cell, biological to physiological level. And as far as I know, no one has ever, never told me one. So I think, Peter, we need better information under these sort of lower doses, quasi intermittent with a feeding cycle to understand the answer to your question. And Matt alluded to this before, everyone looks at S6 kinase and its substrate S6 phospho S6. There's not that much evidence it matters so much for lifespan. There's some. What are the real relevant targets there?

Let me just add on because I think a lot of what you said, David, is spot on and maybe worse, extending a little bit. So this last point about which substrates, there's very little information about other MTORC1 substrates or MTORC2 substrates. In the context of this question of when you look across tissues, how much inhibition do you get? And it very likely, as David already mentioned, even rapamycin doesn't affect all of the MTORC1 substrates. And you would expect that that at higher or lower doses the relative effects on different substrates are going to be different. So there have been a few studies looking at S6 kinase and maybe MTOR phosphorylation of itself across tissues in the context of aging. And there are some variations. But I will also say those studies have differed from each other because the way the experiments were done were the mice fasted and refed before you measured mtorc activity, which affects mtorc activity wasn't the same across the study. So the real answer is we don't know this. Brain penetration question, again, as David, I think correctly noted, there's disagreements out there about how effectively does rapamycin cross the blood brain barrier? How much rapamycin do you need to get inhibition of MTORC1 in the brain? What I can tell you from our own studies is certainly at higher doses, and I think this matches what you've seen, David, is that we see potent inhibition of MTOR complex 1 in the brain after repeated dosing at higher doses where we're using IP injection. We haven't really compared this to lower doses where the rapamycin is in the food. The one thing I'll say is, and this is speculation, but I think it's reasonable speculation, we know that with age there is a decline in the function of the blood brain barrier, that many molecules penetrate the brain better in older animals compared to younger animals. I speculate that that's probably true with rapamycin. And so in the context of aging, it wouldn't surprise me if you actually get better penetration of rapamycin across the blood brain barrier in aged animals and in aged people potentially. But I don't know of any real data to support that. So these are all questions that I think need answers and there just isn't much out there right now.

Couple of questions and then a follow up comment. What's the size of rapamycin? How physically large is it?

It's almost exactly a thousand daltons. In the world of small molecules, it's a big small molecule. A hydrogen atom is a dalton. So it's about 1,000 hydrogen atoms sort of weight most small molecules more in the 2 to 3 to 400 range. This is big.

What's the size at which you can easily traverse the blood brain barrier?

I think here this is not as relevant because I think it's a very, very lipophilic molecule.

It's more about the solubility than size.

Yeah, I almost see like a lot of it gets trapped in the membrane. You almost sort of need to sort of push it it through and you know, and the brain has a lot of things like myelin, which are all very lipophil. So I think there's almost like a sink of sort of trapping rapamycin in places that maybe it's not so effective.

The only anecdote I would add here, and I don't know if it means anything, I would love to have a crystal ball that says in five years we'll have a better answer to this particular question. But there's a biomarker called C2N. I don't know if you guys are familiar with it. It's a biomarker that we use clinically in humans, of course, to look for amyloid in the serum, but it's very highly correlated with amyloid in the cns, and it's very highly correlated with amyloid PET scans. So obviously, in patients who are high risk for Alzheimer's disease, if they're in a clinical trial, you might be able to justify amyloid PET or lumbar punctures to look for amyloid in the cerebral spinal fluid. But not only does that come with the case of a lot of radiation and potential morbidity, respectively, for those procedures, it's simply not practical if you're clinically practicing medicine. So this C2N assay, which was approved a couple of years ago, has become a really important part of how we manage risk in our high risk patients. And this is very anecdotal, but for our very high risk patients who are showing amyloid already in the plasma, I believe we have put, put two of them on intermittent rapamycin. So anywhere from 5 to 8 milligrams once a week. And in both cases, the C2N score has improved, meaning every three months when we are checking the amyloid concentration, it's going down. There are 10 leaps of faith you'd have to take there. Does that mean the amyloid is going down in the cns? I don't have to spell that out to anybody who's reasonably thoughtful. But I think your point, David, about by definition, these patients are aging, so maybe their cns, their blood brain barrier is not as robust, even though that's a very low and clearly infrequent dose of rapamycin. Maybe it is making its way into where it matters. Alternatively, it may not be making a difference where it matters, and it may simply only be making a difference in the periphery, where presumably it doesn't matter. So there's just a lot here.

Let me stop you there, because I actually want to present a different hypothesis, which is that it's actually the periphery that may matter for the brain. And there's two lines of evidence that I can point to that might support this. One is, you know, we've worked for many years in my lab in a mouse model of childhood mitochondrial disease called least syndrome. It's a complex one deficiency in the mitochondria, but it's a brain disease, so it causes neurodegeneration and lesions in very specific regions of the brain. So we did do an experiment along the lines of what David was asking about in the context of longevity, which hasn't been done where we knocked down mtor complex 1. This was in the case of an S6 kinase knockout in different tissues. And I expected it would be the brain specific knockout that would lead to rescue of the disease. Turns out it didn't at all. It was the liver specific knockout that led to partial rescue of the disease. So there could be a tissue signaling piece, and that could be metabolic. You could imagine inhibiting MTOR complex 1 in the liver would lead to systemic metabolic effects. So I think that's a case in point where you can get effects on a brain disorder. I'm not at all saying the mechanism there is the same as neurodegeneration and aging, but you can get an effect on a brain disorder from inhibiting mtor complex one in the liver. The other thing that I think is super interesting, and there's accumulating compelling data that systemic immune dysregulation drives dysfunction in many parts of the body, including the brain. And in fact, with age, concomitant with the breakdown in the blood brain barrier, you actually may see higher penetration of peripheral activated immune cells into the brain, and that's driving some of the inflammation in the brain. You could easily imagine. And again, this is total speculation, But I think it's plausible that this is at least partly right. Could easily imagine Rapamycin's effects on the peripheral immune system Would then reduce the transfer of peripheral immune cells to the brain, or at least inflammation caused by those immune cells. So it would not shock me at all if you don't really need to get high levels of rapamycin or high levels of MTORC1 inhibition in the brain to derive some of these benefits that people have seen, at least in laboratory.

Animals and our rationale for this, because of course, someone listening to this would be understandably thinking, what the hell are these guys doing? Why would you be giving people rapamycin when you have no idea if it works? And why would you be doing it in somebody with Elevated amounts of amyloid beta. I think part of it is just the hypothesis, which is, look, we pretty much know that there is no meaningful treatment for this condition. And we also know that once you've exhausted all lifestyle measures around treating people with MCI mild cognitive impairment, you're not going to rescue everyone. And when you understand these, these potential improvements specifically around inflammation and autophagy, we can debate the relative importance of each of these. We didn't talk about senescent cells. Let's also come back to that in a moment. It makes sense that this inhibition could have an effect. And I think your point, Matt, is an excellent one that I hadn't really considered truthfully, other than just through broad reduction in inflammation. But you're right, we could be thinking about this through the lens of less PBMC activity in the periphery should improve it. And Alzheimer's disease is a very complicated disease with multiple pathways. There are these very lipid dependent pathways and there's a lipid type of Alzheimer's disease, there's a really inflammatory type of Alzheimer's disease. I think all of this basically speaks, hopefully screams towards more clinical research being done. This gets to a broader point. We've already alluded to the incredibly slow timeline for rapamycin's transition into humans. And the net result of that was a drug that was not a profitable drug, presumably for Pfizer for very long. And as a result of that, there has been a relative lack of interest in studying rapamycin and instead an interest in looking at other drugs. Let's talk about one of them now. So Everolimus, which I believe at the time was part of the Novartis portfolio, is that correct?

I think so, yeah.

How does Everolimus differ from rapamycin? Before we get into talking about one of the more important studies in humans.

I don't quite remember, but it's a small modification. There was a methyl group on rapamycin. Ironically, the original patents on rapamycin did a very poor job of covering obvious derivatives of rapamycin. I was involved in some intellectual property cases is in the patents. They talk about rapamycins and Wyeth, who owned those patents, Wyeth, Iris, that Pfizer eventually bought, was trying to make the argument that that covered a lot of these derivatives. Eventually it was ruled that that was not the case. And so therefore a lot of these so called rapalogs, these derivatives are actually quite simple derivatives of rapamycin that almost many chemists would come up with. And Everolimus is one of those since then there's been more sophisticated variations. But eberolimus is, I think, Matt, a simple variant, right?

I think you're right. And again, maybe just for context, correct me if I'm wrong, David, but I think we can say that there are these classes of what people call Rapalogs which are all going to be chemical derivatives of rapamycin, but biochemically they work by a pretty similar mechanism. They all bind fkbp12 and then it's that complex that inhibits mtor complex one. And I think the real differences are more around bioavailability, maybe tissue distribution and how long the drug lasts before it gets metabolized. I think all of these things are broken down by cytochrome P450 enzymes. And so you're going to get differences in peak and trough levels based on the bioavailability and clearance and then maybe some differences in tissue distribution. But I think those are the primary things that differentiate the Rapalogs. Biochemically, I think they're all pretty similar.

I tend to view them, and certainly in cells and culture they act identically. I think from a biochemical point of view at least, the original Rapalogs were pretty much identical. And in many cases, frankly, I don't want this to sound pejorative. I think they were patent plays. I think they were ways to try to get a new chemical entity that then had a longer life than rapamycin. I think even Wyeth Iris did that. They had a molecule, I think CCI779 I think was its name, which was a simple rapamycin derivative. And a lot of their cancer studies they used that molecule instead of rapamycin because as we spoke, rapamycin basically became off patent very early.

It's interesting, by the way, how expensive it still is. Even as a generic drug. It's still a comically expensive drug, but it speaks to probably the lack of alternatives. So we alluded to something that happened that was really remarkable, if I'm not mistaken. I think it was even April of 2009. I kind of remember this pretty well. Fast forward five years, five and a half years, I suppose. It's December of 2024. I didn't yet know you guys. You and I wouldn't meet David for another year. But that was a very important day because I had already become really obsessed with rapamycin, but was pretty much distraught that it would never make sense to take as a human, that it would never go on to become a human zero protective agent. Because despite how, how impressive all of the data were in all of these animal models. I just couldn't get out of my mind all those transplant patients I was force feeding rapamycin to, like Tic Tacs and Chiclets. And I was just like, hey, this can't be a good thing if you're in the business of living longer. And if it wasn't literally the day before Christmas, if my memory serves me correctly, I got an embargoed copy of a paper by Joan Manic, Lloyd Clickstein and others that seem to at least challenge the very foundation of that. And of course, Matt, you already made a lot of good points about this, which is that thinking about rapamycin might have been a bit premature. So either one of you guys, why don't you walk us through the study in the Australian senior citizens that I think for many people was, I don't know, for me at least a huge turning point in how we thought about this drug.

All I'll say to me is that that paper, the Joe Manic paper with rejuvenation of the immune system, I think will be seen in the aging field, is certainly a milestone paper along with the ITP paper. As far as I know, it's really the first where you actually rejuvenate some organ system in a human being. Right. And so I think her study really was mind blowing. I think Matt can speak more to the details of it.

I would agree completely with that. And we may want to come back and touch on that because I think, think as people are thinking about clinical trial endpoints for jarotherapeutics, that's a perfect case example of a functional endpoint that you can actually do a clinical trial on for FDA approval and show improvement in function and potentially get a drug approved as a Jaro therapeutic. So I think as a conceptual advance, it's important as well. So just to give a little bit of history, there was actually a paper, I think it was 2009, from Pan Zhang's lab in mice that preceded the Joan Manic paper, where they showed that you could treat with rapamycin for, I think, six weeks in that study and rejuvenate the immune function of a mouse. And to me, the one experiment in there that is most compelling is they have a set of mice, I think they were 24 months of age when they started this experiment. And then they had young mice, and the mice got either a flu vaccine or no vaccine. And then they waited, and then they gave them what would be a lethal dose of influenza if they hadn't been vaccinated in the aged mice, they either got rapamycin for six weeks or they didn't. And so if you're a young mouse and you don't get a vaccine and you get this dose of influenza, there's 100% mortality within, I think it was eight days. That makes sense, right? No vaccine, you're not protected against the influenza. If you're a young mouse that got the vaccine, 100% protection. So that again makes sense. It's a control. If you're an old mouse, no rapamycin, you get a vaccine. Only 30% of the mice actually were protected. So this is showing you the impact of just normal biological aging on the ability to respond to a vaccine. In mice, it's about 70% of the time you don't respond to the vaccine and you die than if you get a subsequent influenza infection. Interesting parallels to humans as we've learned over the last four or five years. The cool thing in that study was if the mice got six weeks of rapamycin treatment before the vaccine, they were then 100% protected. These are old mice, they're now almost 28 months old. So this is sort of an amazing demonstration of immune rejuvenation in an aged mammal. So I think that study is what really set the stage and allowed Joan and the group from Novartis to be able to move forward and convince the people who had to fund this study that there was a reason to think that MTOR inhibitors might do the same thing in humans. So the design of that human study conceptually is very similar to the mouse study I just, just laid out. Except of course, they didn't give people lethal doses of influenza. But what they did do was they enrolled healthy older people. I think they were over the age of 65 and there was some set of pre existing disease that they would be excluded for. So they were considered relatively healthy for their age and they got either placebo. I think in the first study they tested three different doses of Everolimus. So it wasn't rapamycin. But I think we can just think about it the same as we would rapamycin based on our earlier discussion. So there are a few interesting things here. So they got I think everolimus for six weeks or a placebo, and they got either.

It was 5 milligrams once a week, 20 milligrams once a week, and I think it was 1 milligram daily was the third.

That was what I was going to say. So I think between the two of us were close, if not Spot on. Yeah. So that was for six weeks, and then they gave a flu vaccine, and then they looked at antibody titers. I don't know if it was this study or a later one where they looked at viral gene expression as well. And then also subsequent infections over the next, I don't know, six or 12 months or something like that. Respiratory tract infections. So the first paper, what the first paper showed was, I think, pretty convincing data that at least at the 5 milligrams once a week and 1 milligram daily dose, there was a boost in response to the vaccine as measured by antibody titers. So that supported the idea that similar to what had been shown in mice, you could in fact, to some extent rejuvenate the ability of the aged immune system in humans to respond to a vaccine with transient dosing with a rapamycin derivative. Everolimus, in this case. The other thing, though, that I think was super important about that paper was it was pretty large. I mean, not huge. It wasn't like a phase three, but there were hundreds of people.

Yeah, I think it was about 80 per arm. Arm.

Okay. So hundreds of people in this study who got everolimus, who didn't have an organ transplant and weren't taking other immunosuppressants. And the side effect profile, at least in the 5mgs once a week group, was essentially no different than placebo. And so I think that study started and it's been slow because there's still a perception that rapamycin has a lot of bad side effects. But that started at least some people in the community thinking, and this, I think, is getting what you were were talking about, Peter, maybe it is possible that lower doses of arapolog in relatively healthy older adults could be well tolerated. And maybe this idea that as a gerotherapeutic we might be able to give rapamycin to older people, maybe it's not so crazy. I think that's one of the important aspects of the study, independent of the potential immune rejuvenating effects, which I don't want to minimize because that's hugely important. I actually think both of these things are. Are important things that that study set the stage for.

And I think from that study comes a word certainly wasn't coined in that study, but in my mind at least it went from, we shouldn't think of this as an immune suppressant. We should think of it as an immune modulator. And that was a clear example of how you take at least an aged immune system and make it more robust and it might be. In fact, it very likely is the case that you can also suppress the immune system. Interestingly, these are the same parts of the immune system. I mean, immune system. We talk about it with one word. It's a very complicated system. But it is the same immune system that is there to fight a virus that is also there to reject an organ. I mean, these are not just T cells, but this is part of the cellular immune system. So that also, I think, is a very interesting footnote to the story.

I was living through this at Hopkins, the age of the immunosuppressants. I mean, remember how miraculous cyclosporine seems? And then FK506 and rapamycin to some extent got caught up in being this generic sort of immunosuppressant. But the truth is, when you looked at the data in cells and culture, it's actually not so easy to inhibit in some of those immune activation assays in culture. Rapamycin is pretty weak. If you look at the data in mice, it never looked like FK506 and cyclosporine, but it got caught up with that name because that was sort of that revolution that was happening. And I think, as you and Matt have said, said that has sort of persisted, but it never kind of looked.

I don't think any patients are using rapamycin today, with the exception of legacy patients. In other words, I've talked to many transplant surgeons and said, is rapamycin anywhere in your immunosuppressive regimen? And I've never heard anybody say yes. Now, obviously there's gonna be somebody listening to this who still uses it, but I think there are patients who still take it who received transplants 25 years ago, and it's part of their regimen and it's working for them, and no one's willing to shift it. But I think you're right. And one part of the story I've never familiarized myself with is the literature that led to its approval for transplant patients in 1999. You would be more familiar with that, of course, than I am.

Yeah, I don't quite remember, but I remember this study that people who take immunosuppressants chronically have higher rates of certain types of cancer, which of course makes sense. Rapamycin does not. And it was justified at the time that the reason rapamycin did not is because it itself has anti cancer properties. Now, the alternative is that it doesn't actually impact the immune system in the way that the other ones do to cause that. And that's never actually been quite resolved. I think all of you are very right to say that this is not a traditional immunosuppressant in any way, but that name has been attached to it. And people say, yeah, I don't want to get infections by taking rapamycin. And I think there's almost no evidence that there's actually an increase in infections at all.

Let me ask you guys a question. We're going to come back to talking about broader topics, but do you believe that if you could look at the epigenome of the T cells in those patients in the mannic clickstein study, do you believe that you would see a change in the methylation pattern pre and post rapamycin?

Absolutely. But I think what you're really asking is, would we see a change in the methylation pattern that is what people are calling a reversal of biological aging.

It's exactly where I'm going. Which is, given our shared interest in that topic as well, which is, is rapamycin effectively doing that? Is it rewriting the epigenome? Is it undoing some of the aging of the T cell and is it writing that code via methylation onto the epigenome?

I don't have a strong enough feeling to make a strong prediction there. Like I said, there's no question you will see a change in the epigenome, but that's kind of just big that you do to a cell is going to affect the epigenome. I'm less convinced that these epigenetic clocks are really measuring from a biological aging perspective what some people think they're measuring. I don't have such a strong feeling that rapamycin would reverse what people are calling the epigenetic aging clock universally. I think in some contexts it will. In T cells in particular. I don't know. I mean, it's a really interesting question. First of all, what are the canonical age related epigenetic changes in T cells and how closely are those to the functional declines that we see with T cells that go along with aging? I don't think that's really been carefully fleshed out. And so I guess I'm just less convinced what the epigenetic clocks are actually measuring to be able to say with any level of confidence that rapamycin is going to reverse it.

No, I think the current versions of the clocks are not measuring anything that's of interest, truthfully. But I still wonder if we just don't have the technology yet to actually read this at CPG resolution and therefore we don't really Know what the heck is going on when we use these crappy micro arrays to read these things, when we're sort of averaging out methylation patterns, I think it's like trying to play the piano with mittens on. It's totally unhelpful. But if you can take the mittens off and put your fingers on, it's a different sport.

To get to Matt's point, we had actually tried to look at the impact of rapamycin on specific methylation patterns, not only on the DNA itself, but also on histones and using a variety of different tools. And the truth is we never published this because we almost found nothing specific. And all the impacts really were from the cell cycle delay. Once you sort of normalize that away, you couldn't say, hey, mtor inhibitions regulating K27 this or that. There wasn't there. That signal wasn't there. It really was an impact of delay. And so I agree with Matt, you're going to see impact.

But why, David? So that's very interesting, but how would that explain what we just saw, that in six weeks, which is nothing in the span of a person's lifetime, six weeks of inhibiting mtor. And again, let's do it in the mouse experiment, because that's so much more dramatic. And now, admittedly, six weeks might be analogous to a year or so in a human's life, but in a relatively short period of time, you have a log function change in the immune system of the older mouse. It's hard for me to understand how that could be explained by something that is just cell cycle specific and not a fundamental rewriting of the genetic code of that cell. Again, I could be just completely naive here, but it seems so profound.

Peter, this gets to the fundamental question here, is what is wrong with the age lymphocytes and what does rapamycin do to them to fix the that? And so what I'm telling you is that cells in culture, we always imagine there's a signal transduction pathway from MTORC1 to a specific epigenetic change. What I can tell you is we found no evidence for that. Now, that inhibition of MTOR in a living system with lymphocytes that are impacted by many different signals coming at them will acquire a different state that's reflected epigenetically. And I pretty much think that's what Matt said, a cell state. Rick Young always used to say epigenetics is the setting of the state, not the thing that gave you that state at the beginning. Right. And this is an important distinction so that those cells will be in a different state. But how they got to that state, which in essence is what we're asking, we don't know. So I completely agree with you, Peter. They're in a different state. What I am saying is that the evidence, at least in our systems in cells and culture of a specific signal transduction pathway way, such as the one we can define from MTORC1 to the autophagy machinery, where there's a whole relay of proteins that we can get to the structural level, I don't know and found no evidence for one to the epigenetic state.

Let me just add a couple of thoughts here. So one is if you think about go back to the hallmarks of aging, which there used to be 9, now there's 12 epigenetic changes is only one of the hallmarks of aging. And you can find evidence in the literature that rapamycin impacts all 1212 hallmarks of aging. But the link between rapamycin and epigenetics is much weaker than some of the other hallmarks, like mitochondrial dysfunction, proteostasis, nutrient signaling. So it's not as obvious, but I think rapamycin is going to impact epigenetic changes with aging. And this gets back to the complexity of the downstream part, which we haven't even touched on all the different things that MTOR complex 1 and MTOR complex 2 regulate. Talking specifically about the immune system, though, I think one way to think about this, and again I'm speculating a little bit. I think again there's reason to think this is at least conceptually partly the case. We know that with aging it's not that immune function declines globally. There is a decline in the ability of the immune system to respond to certain challenges and hyperactivation of the immune system towards other challenges it shouldn't respond to. That's why we get so much autoimmunity with aging or this sterile inflammation. Just from a very simplistic conceptual perspective, you could imagine that one of the things rapamycin is potently doing is knocking down this hyperactivation. And this is something I wanted to mention, but we didn't talk about. In both the Mannix study and the Panzheng study, the vaccine was given after the transient treatment with rapamycin was stopped. I would really like to know what happens if those mice or people were continuing to receive rapamycin when they got the vaccination. But in the context of that design, you could easily imagine six weeks of rapamycin is enough to knock down chronic Sterile inflammation to the point where you have a resetting of immune function which then allows the immune system to appropriately respond in a way that functionally is like a young immune system to a vaccine. So I think you don't even have to say that this is fundamentally an epigenetic phenomenon to account for the observation. Functionally, we can rejuvenate the ability of the immune system to respond to a vaccine and potentially protect against a bunch of other types of infections going forward. I also think that's how you can sort of account for the persistent effects that we see with rapamycin treatment transiently in mice, in other places like the heart or the brain or the ovaries or the oral cavity, where we know that six to 12 weeks of treatment is enough to apparently functionally rejuvenate those tissues and organs, and that that effect persists for some period of time going forward after you stop the treatment.

Which begs a question, to cycle or not to cycle. So Matt, you wrote or co authored a paper that came out earlier this year that was a survey, not an experiment, but a survey that looked at over 300 users of rapamycin. So this is a bunch of people who are clearly using rapamycin off lately label, which is a completely legal thing to do. It just means that there is no indication for its use. And you compared them to a group of people you tried your best to match. Nearly 200, if I recall, who were hopefully as similar as possible in terms of their health consciousness, which would be an obvious confounder, but who are not rapamycin users. Can you give us some of the highlights of what that survey discovered?

So, yeah, I mean, I think you described the study pretty well and I think it's important to be cognizant of all of the limitations that go along with the study like that. Because it was all self reported, all survey based. We got in some ways lucky in the sense that the two populations that what we would call the users and the non users appear to be pretty similar in terms of demographics and lifestyle habits and as you said, seem to be similarly health conscious. It's clearly a biased cohort. So if you look at the responses that the individuals gave to the surveys, I don't have it sitting in front of me. But in terms of lifestyle factors, this is a population that is not normal for what we would think of as middle America. Much more health conscious than I think we would see if we had a swath of just middle America. But for what it's worth, they seem to be pretty similar and so there were a few take homes from that study. I think the biggest take home for me is that there really was no evidence when you looked between the people who were using rapamycin off label and the people who'd never used rapamycins for significant side effects of any sense other than mouth sores. One of the surveys was a list of, I think 30 or 40 potentially common side effects that have been associated with rapamycin or with other drugs. And the question was very simple for people who'd been using rapamycin for at least three months. Have you experienced any of these in the past three months? And then for people who never used rapamycin, same question. The only thing that came out is statistically significantly more common in the rapamycin users was mouth sores. And that makes perfect sense. That's the most common side effect that organ transplant patients experience. And lots and lots of people who've used. I think, Peter, you've talked about your experience with mouth sores.

I have a wicked one at the base of my tongue right now that I almost burnt before this podcast.

So in a sense that's a nice positive control.

I was just about to say it's my only biomarker that I know that I'm getting high quality rapamycin.

Right. So in a sense, it's nice to see that. And it's interesting. That was the only thing.

What's the approximate frequency? Because I think in the manic study it was surprisingly low at 5 milligrams weekly. It was like 15%.

Yeah, I think it was like 15% in hours as well. Yeah, I think that's exactly what it was actually. So 15ish percent of people reported mouth sores.

Any idea why this is happening? Is this believed to be immune mediated?

I don't have a good explanation, David, do you?

So I have a couple thoughts on this. I think first, you're obviously not looking at the rest of your GI tract, so you don't really know what the potential sores are elsewhere. I mean, these are epithelia that are turning over in a couple of days. And we know from many studies, genetic as well as pharmacological, that rapamycin tends to impact hyper proliferative cells. If you look at, for example, the impact of MTOR hypomorphs in brain development, it tends to be when you make the telencephalon, the cortex, where there's massive bursts of proliferation. Lymphocytes, as we talked about, divide every eight hours. That's pretty atypical for A mammalian cell, I would argue it's sort of epithelia proliferating fast and you're slowing it down and perhaps losing barrier function.

We don't see side effects at the fingernails and the hair, which are other places where you would expect to see it, at least based on chemotherapy. Traditionally, yeah.

Although there are studies arguing, for example, I know we've even done this, if you give high dose rapamycin before you give some chemotherapy, you can actually, for example, prevent some of the hair loss you in mice when you give chemotherapy. But then as soon as you remove it, it's clear that you just arrested the cells and then they all sort of fall out afterwards. Right. Sort of in a block. One thing, Peter, that I've always told many people in the pharma world for the mouth sores, which I know trouble people a lot. I've never taken rabamycin, but I know it can be pretty bad. Why don't people do FK506 mouthwashes? I don't get this. Because all you need to do is occupy. Stuart Schreiber showed this, I don't know, ages ago. If you occupy the FKBP of FK506, Rapamycin has nothing to act on in your mouth. And you'll prevent this because as far As I know, FK506 does not do this. And so you just need to occupy or even with a benign, a rapamycin like molecule, all you need is an FKBP binder to sop up the binding sites that rapamycin would use.

It probably depends on the frequency with which you do it and what FK506 tastes like.

Like, sure.

But if the mouth sores are that bad, there are rapamycin FK6 analogs. They're completely inert. They simply bind to FKBP, but they can't then target calcineura in the case of FK506 or MTOR in the case of rapamycin. All I'm saying is you just need to tie up your fkbp.

Yeah, no, it's interesting. A little FK paste.

Yeah, interesting experiment and I think you're probably right. But that does make the assumption that the mouth sores are actually caused by inhibition of MTOR in those cells inside the mouth. And I don't think we formally know that at this point.

Completely agree. We don't know that that would be.

The experiment to help elucidate that or.

A more interesting experiment, and this is something we would love to do, is whether rapamycin toothpaste or rapamycin mouthwash, or something like that, specifically delivered to the oral cavity. Is that sufficient to get some of the benefits that we've shown in mice from systemic rapamycin treatment on periodontal disease, gingival inflammation, and bone growth around the teeth? So that's again, a tangent from what we were talking about, but I think super interesting and unexplored.

Talk to me about any of the immune stuff that you saw, because you happened to run this survey during COVID What did you learn there?

So, first, to go back to the side effects. There were other side effects that were statistically different between the groups, but they were all the other direction, lower. The people who had been taking rapamycin, those included, included things like abdominal cramps. It's harder to really develop many hypotheses around. The ones I thought were interesting were depression and anxiety. And there's a whole growing body of literature on the role of MTOR and inhibition of MTOR in various types of neurocognitive behavioral aspects. And so it makes me wonder if that actually might be real, that to some extent in some people, rapamycin could actually have some. What in this case appear to be beneficial effects, may not always be beneficial effects on things like depression and anxiety. So I thought that piece was interesting and certainly worthy of further study. And I know there are some people working with rapamycin, sometimes in the context of ketamine, for things like depression, chronic pain. So I think there's a lot of interesting biology there that hasn't really been explored.

Can you say more about that, Matt? Because I was just about to ask you about what is ketamine doing to mtor?

I thought it was the opposite. Guys, I thought rapamycin caused depression, right? I thought in other types of trials, rapamycin depression was one of the side effects. And certainly the ketamine study argued that as well.

Right. Because ketamine is activating MTOR in the cns, isn't it?

That's right. The data I'm familiar with and the clinical use that I'm familiar with is the context of rapamycin actually in combination with ketamine, enhancing the effects of ketamine both in terms of magnitude and how long they last. In other words, when you combine rapamycin with ketamine, you can sometimes go to a lower dose and reduce the frequency at which patients are using ketamine. Although, again, I think a lot of this is not published. There are at least a couple of studies that have showed a potentiating combination effect of rapamycin. With ketamine in, I think, patients with severe depression, But I don't remember for sure off the top of my head. I've talked to psychiatrists who are using this combination who at least give anecdotal reports of pretty potent outcomes in some patients who have severe chronic pain from combining rapamycin with ketamine. So again, I think it's pretty early. A lot of this is being done off label and is not being written up the way we would like it to be reported in the literature to release so people can learn from each other. But there's absolutely people using that combination now in clinical practice.

That's interesting because I think the initial, I think it was from Dumont at Yale. I think the original ketamine study argued that rapamycin blocked the effect of ketamine. And that was partly the argument that MTOR was involved. I think I recall also, Matt, what you're saying, that there's some discrepancy there and it might be blood brain barrier access, it might be things like this that are quite different and very dose dependent.

Sounds like we need to go back to that original study and make sure we all. We're on the same page. So all I can tell you is I know from conversations with people who are actually using this now that there are people using the combination of rapamycin with ketamine and at least anecdotally sometimes reporting pretty significant changes in outcomes.

And that ketamine is intranasal, intravenous, intramuscular. Does it matter?

I don't know. Outside my area of expertise.

Let's go back to the survey. The other thing that I remember jumping out at me was, and again, lots of confounders here. If you have a healthier population who's more health conscious, and that's why they're taking rapid, because they're literally at the periphery of what one would do. That could easily explain the observation that they got Covid less and when they got it, they were less impacted by it.

Yeah. So let me tell you what we observed in the day data with all the caveats that there are around the way the study was designed and carried out. So within again, two populations, people who had ever used rapamycin, they're all in the rapamycin user group. People who had never used rapamycin, they're in the non user group. But when you look within the rapamycin user group, we actually had three categories of people in the context of COVID 19 infection, some people didn't start taking rapamycin until after they had had their COVID 19 infection, some people took it before, but not after or not during. And then there were people who took it continuously throughout. And so we tried to group them that way and look at if there were any differences between the groups. So first of all, no difference in frequency of infection that was significant. So there's no reason to believe, based on our data, that rapamycin impacted the likelihood that somebody would get a positive COVID 19 result. This is self reported. So we asked people to confirm that this was a positive result from a test. But we're going by what they told us. We don't have any laboratory confirmation. So the interesting thing was that the people who took rapamycin after they got their COVID 19 infection looked just like the people who never took rapamycin. That makes sense, they shouldn't. And we were looking at two things. Severity of infection, again self reported as mild, moderate or severe. And we had specific criteria for or length of symptoms and hospitalization for each of those groups. And then self reported long Covid as in experiencing ongoing symptoms of COVID after a three month period. So no difference between people who started taking rapamycin after their infection and non users. No difference between people who took rapamycin before their infection but stopped taking it. Big difference, at least statistically significant between people who took rapamycin throughout and all of the other groups where people who took rapamycin throughout had lower severity of infection. And the numbers were really small. So I don't want to make too much of it, but statistically significantly less likelihood of reporting symptoms associated with long Covid. So it's at least, I think suggestive of the idea that rapamycin continuous use throughout the period of infection and resolution of symptoms, it may be associated with a lower likelihood of severity of outcome and lower likelihood of long Covid. And again, I think that might make sense in the context of at least how at a crude level, we think long Covid in particular is working and severe COVID infections, which is there's this hyper inflammatory or chronic inflammatory response, it kind of makes sense that rapamycin use may have benefits in the context of that prolonged inflammation or hyperinflammatory response. So that might explain what we saw in the data. But again, I think it's just suggestive and worthy of potentially future work to really disentangle. And I will say I don't think there's any reason to think this is specific to COVID 19. This may be a general property of rapamycin for a bunch of different types of at least viral infections.

David, you mentioned a moment ago you've never taken rapamycin. Obviously Matt and I have of say a little bit more about that. Obviously you're one of the most knowledgeable people on this topic. I think it is perhaps somewhat telling and maybe important for folks who are out there considering it to understand why your decision has been not to take it.

I always used to joke that when I was purifying mtor, I got a huge dosing and given that early exposure is better, I got the benefit then I never wore gloves and it's a powder. I remember it would get into my nose and stuff. So I've snorted rapamycin.

Fair point.

Inadvertently. So I did get a dose at the time. Time now, you know, Peter, it isn't such a willful thing. It's more that it takes some effort to go and actually do it. But I do wonder, and you and I have had this discussion if you eat okay and you do exercise, if rapamycin is a memetic to some extent of a healthy diet. I know it's more complicated than that, but if we call it that, are you getting that extra benefit? Right. At the doses in particular that we're talking about? And so. So that would be my biggest question. It wouldn't be am I afraid of it? I'm not. But will it actually do anything?

But isn't there sort of a hedging or a pascal's wager which is as long as you could convince yourself that it's not harmful, would the worst thing you're doing is wasting a lot of money because it ain't cheap.

So I agree. But then that's where the laziness factor comes in and sort of figuring out how to do it and stuff. But what I would really like to know, and this is what I'd like to study in the future, is getting back to. I think Matt, you've mentioned it. Peter, you have this cyclical nature, right? I'm much more interested in sort of a. Because what can't I do? If I starve myself, what happens? My body synthesizes certain nutrients, break downs other things to release them. And in fact, when you look at the metabolic state of a mouse that you've starved, the levels in the blood are pretty similar. So I can't, through dietary interventions, starve a cell of nutrients like I can in a dish. I can't. The body fights that and of course eventually you run out of stores and you die. But in a normal type of starvation situation, so what I'm much more curious about is can I use rapamycin or other MTOR modulators, perhaps God forbid, even catalytic inhibitors, to take that system to a state that I cannot simply do with a dietary intervention whatsoever. And obviously that is not sustainable in any chronic way. We know that if you give a catalytic inhibitor to a mouse, you can actually kill a mouse fairly easily. It's actually hard to kill a mouse with rapamycology Mycin.

Can you remind folks again the difference between an allosteric and a catalytic inhibitor and what that actually is doing in the case of mtor?

So the allosteric inhibitor, rapamycin and derivatives is going to do this partial inhibition.

Of MTORC1, the rock that partially obstructs the cave entrance.

Exactly, the partial block, and also partially inhibit MTOR 2. And there's going to be perhaps some tissue specificity, some kinetic differences, a catalytic inhibitor, which is basically a molecule that will compete with ATP, which is what MTOR uses to do all its business, that will obliterate MTORC1 and MTORC2 activity, certainly when given at the right doses and in our hands is highly toxic to cells and to organisms. Again, we have misdosed by mistake. Catalytic inhibitors in a mouse and a mouse will drop dead.

When you say drop dead, are you talking about the same way where mitochondrial inhibitors like cyanide, which immediately cease respiration, will kill an animal with insects?

No, it'll take usually a couple hours. The mouse will stop moving, it'll get cold, sometimes it'll have seizures, but it will die.

But still profoundly and acutely toxic.

Profoundly bad, yes, which rapamycin does not do. So clearly one has to be careful of those molecules and the clinical experience has suggested that. Right. These were molecules that were initially thought to be potentially good anti cancer agents. We made some of the first ones and also we're touting it from that. But I think the experience has been that they have lots of side effects. But I've always wondered, can those molecules in a careful way be done to very much impact this system, massively activate autophagy, massively rewire this system, maybe have epigenetic impacts very short, and then come off of that. I'm much more curious about that type of study and potential use because I feel that again, with diet you can get close to rapamycin's impact. Again, this is my personal belief, with some data to support it. But what I know you can't get close to with diet is what a catalytic inhibitor can do.

I think you said that, and I've tried to make this point before, and I think you said it in a way that I've never thought about it, or at least I've never said out loud, which is important. The point is that rapamycin is very different than dietary restriction. They're overlapping, but they have lots of differences. And I think you're right. You can't have the same impact on MTOR systemically in tissues with dietary restriction that you have with rapamycin. The other side of that, though, that's equally important maybe is that dietary restriction does a bunch of other stuff that rapamycin doesn't do. And the potential benefits and negative consequences of all of that other stuff, I think are often not weighed into the equation when people are thinking about diet and comparing it to rapamycin. The catalytic inhibitors, though the point I wanted to make is that there's two. One is most of these catalytic inhibitors are less specific for MTOR than rapamycin, meaning many of them affect other kinases. Not all of them, but many of them do. And there's this whole class of what people call dual kinase inhibitors that hit other kinases. David's shaking his head, so you can tell me why I'm wrong. But there are other proteins that some of these molecules that inhibit MTOR will also inhibit. And RTB101, which we didn't talk about the future, the subsequent studies from Joan at Novartis and then when she went on to restore Bio. There's this other molecule, RTB101, that I think would fall into these ATP competitive MTOR inhibitor class, but it also inhibits other kinases. So the specificity for some of these molecules is less. I don't know if we know in terms of the side effect profile how much of that is due to MTORC1, MTORC2 or other kinases that these molecules inhibit. But I do think it is worth saying, at least in the studies that Joan did at RestoreBio, they did dose people with RTB101 and did not see significant side effects. So you can ask whether they saw significant efficacy. That trial actually was shut down, but it is possible, at least for that molecule to use it clinically at doses where there's some reason to believe there might be some efficacy.

Before letting David chime in, can I just ask a question to clarify that in the RTB101 trial, didn't they combine it with another agent?

They did with Everolimus. So they had two arms, one was the combination and one was RTB one. Hundred one alone. Yeah.

My shaking was that I was agreeing with you. And that study that MANIC did after I was confounded by that study and perplexed because this RTP which they, they 101 they renamed, I think it was NVP103 which was a Novartis molecule that's a dual MTOR PI3 kinase inhibitor and actually a very dirty molecule. I remember being on some advisory panels for Novartis and really not understanding why this molecule even existed. So you're right, the ATP competitive inhibitors are dirtier than rapamycin by far, but not all of them. In fact, Wyeth had made a compound under the guidance of Bob Abraham, who was one of the pioneers in amateur biology, which is exquisite to relatively specific. You can dial out pi3kinase activity of the catalytic inhibitors. But the kinase that was very hard to not also hit was DNA pk, a kinase involved in the DNA damage response. The molecule we made, Torin 1 we never managed to dial out DNA PK, he did. So this Wyeth compound is a beautiful molecule. When Pfizer bought Wyeth they de emphasized it in favor of dual activity inhibitors which again I did not agree with. I do think there are some quite good molecules and that's the molecules that we use, these very hyper specific ones and they are bad news for an.

Animal when you use this gets back to low hanging fruit that hasn't been studied. I would love to see somebody take a panel of all of the known MTOR inhibitors in these different classes and just ask the question if you look in an animal model, what's the relative benefit and side effect profile look like in the context of longevity? I'm confident that at least in worms you will find things that work better than rapamycin because we've already done it. I don't know about in mice, but it seems like a really important question to understand the biology of these MTOR inhibitors in the context of aging to know is rapamycin really best in class or is it just the one that we studied the most? And that seems like a completely unknown to me at this point.

You would just have to guess that it's not best in class in the same way that the first of anything it could always be perfected, right? I mean that would be your guess.

Yeah, absolutely, that would definitely be my guess.

It would be my guess too. But you know the balance between full MTORC1 inhibition, total MTORC2 inhibition, I don't know the answer to that. And one of the reasons I think this hasn't been done is that the catalytic inhibitors are actually very challenging to use. They're very hydrophobic molecules because the catalytic site of MTOR is like a very hydrophobic site. So everyone who independently made these molecules ended up with very greasy molecules that are not easy to dose in a mouse. Very hard to dose. You got to put them in detergents. All these things that the mice don't like either. But I completely agree. But I would do that study, Matt, in an intermittent way. That's the way that I would want to do that, to sort of mimic, make a really strong inhibition of this system and then release and see what happens.

Guys, why do you think that they put forward RTB101? I mean, you made a point a minute ago, David. I mean, it was probably more of a PI3 kinase inhibitor and a dirty one. Exactly like. I also was confused. And the problem is, when that second study came out and it was a null study, it somehow got interpreted as, oh, wait, Everolimus doesn't work. Which, again, there's no scenario under which I would make that interpretation. But help me understand that, because you wrote, if I recall, Matt, you wrote an editorial on this, if I'm remembering correctly.

Right? So there were actually three studies. The study where RTB101 was used alone was actually the third, and that was their pivotal clinical trial. There was a second phase, two in between the 2014 paper and the pivotal, where they used a combination of everolimus with RTB101. And I wasn't in the room, so I don't know exactly what went into the thought process of why use RTB101. I've been told there are probably at least two factors that played in. One was that in cell culture models, there was Some data that RTB101 induced antiviral gene expression. So there was some somewhat plausible biological rationale for the endpoint that they were going after, which was, if I remember correctly, at least for the pivotal, it wasn't so much vaccine response, it was subsequent infections. And so the thought was, if you can both boost vaccine response and enhance resistance to subsequent infections, that might be a combination that was useful. So in the second phase, two, the RTB101 showed a signal. RTB101 +everolimus also showed a signal, but RTB101 alone showed a signal. So the decision was made to go to the pivotal with RTB101 alone. I don't know the rationale for that. You could speculate it Might have something to do with patent life. Right. And IP around longer patent life on RTB 101 clearer path to market. I don't know for sure, but that's what happened. So there was no ever alignment in the pivotal phase three. There are a couple of things about that trial that are worth just mentioning. One is that Everolimus wasn't in there. So the failure of that trial absolutely should not be interpreted as a failure of rapamycin or rapalogs because there was no rapalog in that trial. The other piece though that I think is worth mentioning is that trial was only half completed and the decision was made halfway through to stop the trial because they were not hitting their FDA mandated endpoint, which was patient reported infections, not laboratory confirmed patient reported. So they were not hitting that endpoint and the decision was made to stop the trial halfway through. That was actually November of 2019. I remember I was at a conference with Joan, the Gerontological Society of America conference. When that news came down, I was upset. I'm sure Joan was even more upset. But if you think about where the world was five months later, they might have made a different decision at that point. Point with a drug that could potentially affect vaccine response and subsequent viral infections. Regardless, that's all history. But now Joan did go back and do a subsequent analysis on the data from that half completed phase three. And in fact, in those patients who got the RTB101, there was a significantly lower risk of subsequent infection for certain viruses, among them influenza viruses and coronaviruses. Not COVID 19 because we didn't know about COVID 19 when this was happening. But coronaviruses as a class. The people who'd gotten RTB 101 showed a significantly lower likelihood of a future laboratory confirmed viral infection. So whether that trial was actually a failure. It was a failure in the sense that they didn't get to FDA approval and they shut it down early. Whether it was actually a failure of the drug I think still remains tbd, which is interesting because this wasn't a rapamycin. It was one of these ATP competitive MTOR inhibitors. But I think it's still a little bit unclear if the drug itself actually failed to have an impact on immune function in the population where it was tested.

But it was a very dirty catalytic inhibitor. It impacts multiple pi3 kinases.

Yeah, absolutely. And I mean, that makes it harder from the perspective of even if it did have an impact, how is it working? Is it really through mtor? Is it through some of these other kinases Is it a combination? We don't really know because it is dirty.

So I always worry that the change in sort of use of molecules reflected that. That original study maybe had some issues that we're not aware of. That first study that we talked about as a milestone study was so amazing that why wouldn't you have expanded upon that? I never understood this, but I think what you said makes a lot of sense, Matt.

Yeah, and I don't remember when ever Elimus came off patent, but it's been a few years now. So the patent clock was ticking. I would speculate that it had something to do with the decision.

Decision.

I don't think that's a skeptical point of view. That would be my Occam's razor answer to that question for sure.

But there are now so many rapamycin derivatives. I still imagine you could have picked one up. I'd have to guess, go through a lot of preclinical studies and things.

David, you've talked a lot about the impact of MTOR inhibition. You've already talked about autophagy. We've talked about a reduction of inflammation. We haven't talked a lot about the tamping down of senescent cells and potentially the reduction of the soluble or secretory factors. We have an impact on proteomics. I mean, lots of things are impacted. You tend to think, if I'm not mistaken, that the impact on autophagy is the one that might be most responsible for the life property, the altering benefits we see of that. Do you want to expand on that a little bit? And Matt, I'm kind of curious to hear your point of view on that as well. What do you think? Which pathways, plural, would you rank order as the ones that are driving this? And the reason I'm asking this, I'll tell you where I'm going with the question in advance. It comes down to biomarkers, a topic that the three of us have endlessly, endlessly talked about, which is if we believe this is dominated by autophagy, then we need biomarkers for autophagy. If we believe this is dominated by inflammation, then we need better biomarkers for inflammation. So with that said, I'd like to.

Hear your thoughts when you think of.

Things downstream of MTOR. Right. You can do a PubMed search and find MTOR and rapamycin, literally connect it to anything you want. Why is that? Either there's a specific signaling pathway to that process or there's a simpler explanation, which to me is that MTOR is a major regulator of Protein synthesis. And if you inhibit Mtoranov, particularly if a catalytic inhibitor, you inhibit protein synthesis. So you will impact everything. And so to me, there is the class of downstream molecules that are impacted simply by impacting protein synthesis. I put those in a very sort of broad category that I don't know how to study them or think about them in any kind of specific way. There are then a whole series of processes in which there are truly molecular connections, direct specific molecular connections that MTOR regulates. And as you said, Peter, autophagy, the self eating of cellular components and destruction in the lysosome that came up earlier, where we know that pathway, we know how it regulates protein synthesis, we know how it regulates transcription factors like tfib. So one I think if you had to put in the molecular target of MTOR that's emerged in the last 10, 15 years as very interesting and prominent, it'd be TFAB. It's a transcription factor that what it does is promote the production of these lysosomes, these recycling organelles. And so, yes, Peter, I would put autophagy. If I had to pick one process that is prominently regulated by MTOR and probably accounts for some of its health benefits, I would put autophagy. Part of that is based on a worm study that I'm sure Matt knows better than I do, where they actually tried to look at that they did MTOR inhibition and then they looked at downstream pathways genetically and found the biggest impact of perturbing autophagy. Part of it is based on common sense. It breaks down old things and allows their rejuvenation. The counter, though, to the statement that I just made is that I'm always asked, why does MTOR impact aging and why do other things not? And what I always say is that if you make the analogy of an old house, you can't prevent the aging of an old house or much less rejuvenate an old house by having a plumber, having an electrician. You need a general contractor that brings in all those people, because an old house has everything wrong with it, as we know, or an old car has every part wrong with it. And so I think to some extent, and we almost can't ask the question what is important downstream of mtor? Because the answer is MTOR is special because it does a lot of things, and therefore we can't find one thing that replicates mtor, otherwise we would already found those things. And so I guess, Peter, if you had to pick, I'd say autophagy is the major one. But I think the real answer as to why MTOR and thus rapamycin are special is that MTOR does a lot of stuff and to impact the aging process process, you have to do a lot of stuff. And this is why. It goes back to that question that I always ask. The real aging researchers tell me one thing in a cell that's not broken with aging, and the answer is there isn't one thing that's not broken. And so therefore, to fix or prevent that, you have to act on many processes.

What about you, Matt? Where do you end up on this question?

I don't disagree with anything that David said and I think the House analogy is that's a nice way to think of it because it is the case that MTOR globally regulates a lot of different things and there's probably multiple downstream processes that play a role. And I think what I would say though is that autophagy being an important, maybe the most important, single downstream, directly regulated MTOR process for a bunch of different, broadly speaking, aging effects is not inconsistent with the idea that in a mammal or in a person, and the anti inflammatory effects account for many, maybe most of the functional benefits that we see when we treat an old organism, old animal. I think both of those things can be true. And it's probably the case that the specific effects of MTOR may be different in different contexts, different tissues, different pathologies. So for example, in hypertrophy, the effects of MTOR on cell size may be most important. Important in cancers, the effects of MTOR on the cell cycle may be most important. Those are tied into autophagy. I mean, I don't know that we're going to be successful trying to point to one thing and say that's the most important thing. David's absolutely right though, that in C. Elegans at least it's interesting because it seems to be the case that most if not all of the benefits of inhibiting MTOR can be directly attributed to activation of autophagy. But you go to yeast and it seems to be mostly the effect of effects on global MRNA translation. So again, that may fit with the idea that context is important here for which of these downstream processes are weighted in a relative sense, most importantly for the effects that we see in the context of aging. So that's kind of the way I think about it. But again, I think you and I have talked about this before, Peter. I have very much in the last five to 10 years shifted my thinking, particularly in the context of people and probably in other mammals, towards the anti inflammatory effects and particularly the ability of rapamycin to knock down sterile inflammation in the context of an aged animal. That seems to me to that a lot of the benefits that we see in terms of organ and tissue function can be plausibly traced back to that effect of rapamycin.

Very interesting. I mean, I think that would lead us to think that, boy, if we really wanted to get a better handle on dosing, we would want to look deeper into biomarkers of inflammation. And we do have more that we can look at there. I mean, you know, everybody gets their C reactive protein checked, but we could be looking at a whole suite of interleukins and other cytokines. But when it comes to autophagy, boy, we've got a whole lot of nothing. Probably been three years now since I had a really interesting discussion with Eileen White about this, who's one of the world's experts on this. I don't think I got any argument out of Eileen that we really need. Need a biomarker here, because outside of the lab, when you can afford to take tissue, we don't have much going on. I want to pivot for a sec. And we've done this before, Matt, but again, I think there are people listening to this who maybe haven't heard it. Can you tell us a little bit about what we've learned in rapamycin as we've pivoted to companion animals? So when we talk specifically about cats and dogs. So what is it about cats and dogs that are interesting? Well, first of all, they're a heck of a lot closer to humans than mice are, but they're also not genetically inbred the way mice are. They live in our environment, not a sterile environment. They consume food that probably looks a little bit more like the food we would consume, at least in some cases. So tell us about what you've learned in this study, which has really occupied more than a decade of your research.

So there's two other things I would add about companion animals and dogs in particular, where most of my work has been. But this is also true for Catalyst cats. One is they age more rapidly than people do. So that's super important because that means we can actually measure outcomes of interest in the timeframe that's compatible with a clinical trial. Secondly, they matter. More than 50% of people say that their pet is part of their family. So there's sort of an intrinsic value, I believe, in developing therapies that can improve health span and longevity of companion animals from that perspective.

So just to make sure. Yeah. What you're basically saying is even if we learned nothing about the longevity of humans, this would be a worthwhile pursuit in the way nobody actually cares how long mice live or how long C. Elegans live.

That's exactly right. And I would also say it's ridiculous to think we're going to learn nothing about the biology of aging in humans from studying companion animals. But yes, even if we say that, there's still value in doing these kinds of studies and improving the quality and quantity of life for pets. So I've been involved, as you know, for a while now with a project called, called the Dog Aging Project, which Daniel Promisloe, Kate Creevey and myself started, depending on how you want to do the math, somewhere between seven and 12 years ago with the idea, you know, sort of around what we've already discussed, that there's a good rationale for companion dogs, pet dogs in particular, as a model for the biology of aging, but also to be able to assess rapamycin specifically for its impact on lifespan and health span metrics, because we can actually design a clinical trial, and this is a real clinical trial, double blind, randomized, placebo controlled veterinary clinical trial to answer the question, does rapamycin slow aging, increased lifespan, improve multiple health span metrics in a reasonable timeframe? So we set out to design such a clinical trial. We call it the Test of Rapamycin in Aging Dogs. We've done two shorter term pilot trials, also double blind, placebo controlled, to establish safety, to kind of work out dosing, and then started the larger clinical trial triad a few years ago, which unfortunately coincided with the beginning of COVID 19. So that was challenging, but we've continued to work through that and are making progress. And so this is a trial that will ultimately enroll 580 dogs. Half get placebo, half get rapamycin. The treatment period is three years. We're looking at multiple measures of health span, including cardiac function, neurological function, activity, cognitive function. There's a few others, but I think most importantly, lifespan is the primary endpoint. So with that cohort size, that length of treatment, we are powered to detect a 9% change in lifespan.

Is that remaining lifespan or total lifespan?

That's total lifespan. So life expectancy, it's a bigger number for remaining life expectancy. The reason why we settled on that 9%, as you know, Peter, because you were instrumental in getting us to that point by helping to line up a group of donors who increased the size of the study. The reason why we aligned on that percentage is because that's towards the lower end of what's been reported in mice. And that's in fact what was seen in that 2009 study we talked about earlier, starting treatment in middle age in mice. So again, it's a big question. It's unanswered. Even if rapamycin extends lifespan in dogs and in people, will the magnitude of effect translate? That's a different question we don't know the answer to. But it makes sense to start in the right ballpark in terms of what we think might be a reasonable thing to expect for longevity. So that's why we went with that cohort size. A couple of other things that are maybe worth just mentioning is that the dogs have to be at least 7 years old at the time of randomization and they can't be sick, they can't have any significant pre existing age related disease. And that's important because the vast majority of clinical trials that are done today, whether it's in companion animals or people, are disease specific clinical trials in patients who already have a pre existing disorder. This is a study of normative aging. And so we felt it was important to start with a population that was at least age appropriate in terms of health status. And so that's the study. Dogs are still actively being enrolled.

Any size limitations, Matt?

Yeah, so the dogs have to be between 40 and 110 pounds. And that's for the simple reason that big dogs age faster than small dogs. So again, in order to get the statistical power that we needed, we are working in a population of dogs that are more rapidly ag than a smaller weight body size population.

Can I say one quick thing? You know, you always ask me if I take rapamycin and my friends ask me whether they should take rapamycin because they know you and you take rapamycin. And I always say, well, when Matt Kaeberlin's dog study reads out, if it's positive, I'll take rapamycin.

It's funny you say that, David. I say that to a lot of our patients as well. I say, look again, I have a relatively strong conviction. It's modestly held. It will be a lot more of a strong conviction one way or the other and I'll tighten my grip on it in 2026, which is about the time when we'll have the readout of this study. So yeah, I think a lot of people, Matt, are looking to this study potentially along with the work of Adam Solomon. Maybe we can just touch on that really, really briefly as well as another model.

Let me make a comment on that though. So Even though we're powered for lifespan, that's our primary endpoint. Endpoint. I'm honestly not sure that's the most important endpoint for evaluating potential efficacy of rapamycin in dogs or people. I mean, I think we want to think about this more broadly speaking in the sense that there may be some health span metrics that are particularly and potently positively impacted by rapamycin.

I think people just also want to make sure there's no negative lifespan, though.

Oh, absolutely, I agree. I would be shock. I mean, again, we'll wait till the study's done. I would be shocked if we see a shortening of lifespan from rapamycin treatment. Just given everything that I know to this point in mice and the data we've gotten so far in dogs, it is possible, and I totally understand that reasoning, would surprise the heck out of me if we see any lifespan shortening. Not to say that there aren't side effects from rapamycin, but I don't think there's any reason to believe that it's going to have a negative impact on mortality. Mortality.

We're not seeing lifespan get shorter. We're not seeing an uptick in cancer or something that was unanticipated. So, yeah, if you're neutral to positive on lifespan with these health span benefits in terms of ejection fraction, periodontal disease, things like that, that would probably be sufficient enough reason.

Right, so now the ADAM study. Yeah, so this is a super interesting study in a non human primate called a marmoset. Marmosets are an interesting non human primate model. Model because they aren't as long lived as rhesus monkeys. So rhesus monkeys, I think in captivity will typically live 30 to 40 years. Marmosets, I think this is a little bit of a moving target as people are starting to use marmosets more in captivity. They're learning more about what the actual life expectancy is, but it seems to be towards the, I think low to mid teens, so significantly shorter lifespan in captivity. That makes them an interesting model as a non human primate to study aging. And so Adam, for several years now has had an ongoing marmoset colony in San Antonio, some of whom have been getting rapamycin. And I don't think they've published the data from that study, at least not the lifespan data. So I don't think it's complete, but they've already published some preliminary data for bioavailability, blood levels, some interesting data suggestive. And Adam has talked in meetings about the apparent survival benefits. So Far again, incomplete study where it looks like rapamycin may be having positive survival effects in marmosets. So again, I think if that pans out and we actually see a statistically significant improvement in lifespan from marmosets, that's really important because now it's gotten to the point of a primate, which we don't have data for yet, obviously closer to humans. I think we also, though, have to recognize there's still a pretty big limitation from that study in the sense that it was done in captivity. So there are some questions about rapamycin, particularly. Again, we've already talked about. I don't think any of us believe that rapamycin alone at lower doses is a potent immunosuppressant. But when you're out in the real world and exposed to all sorts of environmental challenges, it may be the case that the effects of rapamycin are going to be somewhat different than what we see in the laboratory. So it's a huge step up, I would say, in the sense of being in non human primates. But it's still got that caveat that it's a laboratory based study.

To me, one of the big issues with the mouse studies in rapamycin is that these are sedentary mice who are getting fat, who are frankly depressed. And so what I always say about your study is the critical aspect is that these are free living animals who presumably are relatively happy. And so the marmoset study sounds exciting, exciting, but it does have that big caveat of potentially more sedentary sad animals and occasions. I actually, frankly hadn't even thought about the potential infectious disease implications of it. But living in the human environment, to me is the key aspect that I'm looking forward to your study about.

I think that's hard to know how important those pieces are, but you're absolutely right. You could imagine they're going to be hugely important. And so, yeah, it's just a difference.

What was the dosing in your study, Matt? Is it 0.1 mg per kg weekly?

No. So we done two. The first one was we tested two doses, 0.1 mg per kg three times a week and 0.05, so half that dose three times a week, and then we went to 0.15 mg once a week, and that's what we're using now. And we could talk about why we made that decision. This is a challenge, challenge. Anytime you're trying to design a clinical trial, there's sort of infinite number of variations on dosing and how you deliver and all of that, we decided to go with that dosing protocol, 0.15 milligrams per kilogram once a week for the large clinical trial, based on the outcomes from the two shorter trials in terms of total dose, so cumulative dose. And we haven't really talked yet about what sort of become popular in the, for lack of a better word, biohacking community, which is this once weekly dosing. But based on Joan's study, the observation that once weekly dosing with Everolimus seemed to give similar efficacy with maybe lower potential side effect risk. And from a pragmatic perspective, because the owners are giving the drug to their dog, we thought that it would be more likely that owners would be able to consistently remember to give the drug to their dogs once a week as opposed to three times a week. And that's speculation, but all of those things kind of weighed in to that decision and I'm hoping it doesn't come back to bite us on the ass. But that's the challenge with designing trials.

And that's where I'm going with it, is if you sort of try to triangulate between the Everolimus study, your studies, Adam's studies and the ITPs, you sort of coalesce around 0.1 milligrams per kilogram weekly for a human, which is kind of putting people put someone my size maybe a bit more, but it's probably like in the 8 to 12 milligram range for someone our size.

I'm not sure actually about that. So this is actually, I was going to say one of my biggest concerns with our dog study is that our dose is too low. We have to go low because you're trying to weigh risk reward, and in people's pets, the tolerance for risk is extremely low. But I am concerned that because we need to be so risk averse that we're having to dose lower than what might be the optimal dose. And my real concern is we're dosing below what would be the dose you would need to see any statistically significant effects. So that's my concern. I don't have any data to point to to suggest that. And actually I have some data to suggest that even at the doses we're using in the two shorter trials, we did see evidence for beneficial effects. But that's kind of the thing that keeps me awake at night when I think about the design of this trial, the mouse studies. Again, I need to go back and really do this conversion. But my recollection of the Mouse dosing was that it actually works out something close to 0.1 mgs per kig per day in people, not per week.

That's absolutely correct. It was 1.4 milligrams per kilogram per day is what the mice were actually given in the itp, which works out to. When you convert that to human dosing, which is. There's a conversion factor, it works out to 0.1 mgs per kg per day is what they were getting if they were humans. So you're right, they were getting much more rapamycin. And yes, that speaks exactly to the concern we have, which is how do you know if you're getting enough? And the only reason I think we may still settle on this weekly dose is we at least saw the positive, positive immune modulation with 5mg a week of everolimus. I mean, that's even less.

All I'll say is that, to me, one of the fundamental differences, from what I hear, and I have no particular expertise, is that the mouse study is really our chronic dosing. And really the best evidence is the manic study for an intermittent dosing having a clinical output that's beneficial. I know there are mouse studies that have done that as well, but in some cases of these larger animals.

Yeah. So I think that's right. I'd say there's two pieces, right? One is the dosing. When you think about, like, daily versus weekly versus every other week, that sort of intermittent dosing. But then there's the question of interval of dosing. How long do you need to dose? And so those are two different variables that I think are both poorly unexplored, even in the mouse studies, to really tease out where you see different benefits or where you get the biggest benefit.

What dose ranges are you seeing in the wild? So when you did that survey, what were you seeing? What percentage of the RAPA users were on weekly doses versus daily doses versus tri weekly? And what was the range of the actual dose?

I think, in a general sense, it was kind of all over the place, but by far the majority were once a week. And among those, the vast majority were 6 milligrams once a week. And I think there's some historical reasons for that. So that's become popular in the online sort of community where people talk to each other.

But where do you think that number comes from?

So I think it's partly from Joan's study. So it's close to Joan's study, which was 5 milligrams once a week of everolimus. I think it's also because many of the people, the first people to start taking rapamycin off label were patients of Alan Green out of New York. And I think that was sort of his standard dose that he put most people on. So I could be wrong about that, but that's my impression is that's kind of how that became popularized, for lack of a better word, within the community. But having said that, there's a lot of variation around that, both in terms of doses that people are taking once a week and then there's a fraction of people who are taking rapamycin daily, usually 1 milligram, 2 milligrams, sometimes for purposes other than purely for aging. So people who have existing autoimmune disorders, sometimes people are taking rapamycin for that. But in general, I would say say among the off label rapamycin users, the majority are once a week and of those, the majority are 6 milligrams. It's kind of a bimodal distribution. There is a group of people around 3 milligrams as well, but lots of variation around that.

What were some of the higher doses you saw for the once weekly folks?

So I think our highest was up close to 20 milligrams once a week. Again, though, it was a little bit difficult to tell how long people had been taking rapamycin at those doses. Again, some people had been taking it. I think the person who's been taking it the longest was many years. I don't know about many, five or six years. So but the majority were six months. These people who reported taking 20 milligrams once a week could be that they just started doing that. And a lot of people, I mean, as you know, right, a lot of these are n of 1 experiments with people who are changing their regimens as they go. So there are some people who are taking, taking 6 milligrams once a week, but they're trying to build it up to some higher dose to see where they start to get side effects. There are a bunch of people who reported taking grapefruit juice with their rapamycin because grapefruit juice will inhibit cytochrome P450s and enhance bioavailability of rapamycin. So, and I think the reason why I say that is both to illustrate the sort of nature of complexity of this population, but also because we know there's going to be genetic variation in uptake of rapamycin and how quickly the drug is metabolized. And so the dose that somebody is taking may or may not really reflect the total bioavailability or the kinetics, trough level, peak level, things like that.

One thing I would caveat, folks who are listening to this, who are themselves taking rapamycin is first of all, it's not a cheap drug. So I think the most competitive pricing you'll find if you're using Goodrx is works out to about $5amilligram gram. Does that sound about right?

Yeah, that's about right.

And so what you're seeing a lot of are these compounding pharmacies that are saying, well, heck, I can just make this for you, I'll make it for you. Instead of giving you rapimmune, which is even more expensive. That's the brand name. Rapimmune is the drug made by Pfizer or just generic Sirolimus. But I would caution people against using any compounded formulations here. Yes, they'll make it a lot cheaper, but you have virtually no guarantee of the purity or the concentration. We're already taking a huge leap of faith with this. We'll have a podcast that covers the ins and outs of compounding pharmacies. I'm not here to suggest they're all bad, but you absolutely want to be able to make sure you have FDA certificates for what you're using and just be careful with the quality control.

I would just add on to that as well, particularly with rapamycin and compounded rapamycin. There is some data out there on compounded rapamycin having essentially no bioavailability if it's not in an an enteric capsule. So this actually goes back to the reason why the ITP took 20 months to start their experiment is rapamycin is unstable at gastric ph. And so if compounded rapamycin is not in an enteric coated capsule, you're essentially going to get zero bioavailability.

This is one where I think you splurge and get either sirolimus or rapamune. Last thing I want to kind of talk about on the potential interesting front. It's the real tragedy of not. I always say this, and Matt, you and I and Dave, we've all talked about this. If I was a billionaire, what would I do? I'd literally just set up a research institute that would fund this type of work with no profit motive because nobody would care to fund this if you were profit driven. But the fact that no one's really looked at the impact of rapamycin on ovarian aging is really frustrating. And by the way, you could also look at the impact of rapamycin on fermatogenesis. But just we're reproducing at a later and later age in life and fertility is such an important part of that, especially as we experience population decline. So anything that we could do to better understand how to preserve the youth of sperm and egg would be really fascinating. I think there is someone looking at this in Brazil and someone looking at this here in the us. I haven't heard enough from it. Maybe you guys know about this more. Do we know anything yet about this?

Yeah. So again, we can go back to the mice and it's pretty clear in mice, female mice, that you can delay or probably even reverse ovarian atrophy up to some point in life with transient rapamycin treatment, actually restore reproductive capacity to sterile female MYOS mice through this sort of treatment. It's interesting though, in male mice it's the opposite. So you actually seem to impair spermatogenesis and potentially induce sterility. It's worth just noting that there may be differences in the male and female reproductive systems there.

Why do you think that is?

I think it goes back to what I mentioned before. If you ask what cells are most impacted by rapamycin in vivo? What is their defining set of characteristics? It's always the most rapidly proliferating. And that I think is what defines spermatogenesis versus oogenesis, which by definition is amongst the slowest processes we have. You need a certain rate of growth anabolism when you're proliferating quickly that you just don't need when you're proliferating slowly and therefore you impact those cells. So I think you're right, Matt. From the studies I've looked at, clearly spermatogenesis, male fertility is negatively in impacted. There's no doubt about that, at least.

Well, on rapamycin, I think there's at least one paper that showed that once the mice come off of rapamycin, there may actually be a preservation of sperm quality in male mice. So again, this gets back to dose and duration and transient versus continuous. I think you're probably right about that. The mechanism potentially just boiling down to.

Cell cycle and the ovarian one. I always wonder, and this is in general what I always wonder about rapamycin and sort of of its potentially anti aging properties is how much is simply a delay because you're slowing the cell cycle and the progression of cells versus a true rejuvenation. And so you mentioned a true rejuvenation and that is very impressive. That's what you're seeing.

So this is not my data and I don't know that the papers have been published yet, but they've been presented at multiple meetings. There's one that just struck me as so profound where you can actually see structural rejuvenation of the ovaries from a atrophied state state to a least at a morphological level appears like true rejuvenation as well as a restoration of fertility.

Of the actual ovary or of the oocyte.

The ovary.

Wow. Which is hard to understand. Right. Because presumably when you're fully atrophied, you have no oocytes left to rejuvenate and.

Well, I don't know about fully. This is where I think it probably depends on how far down the path you've gotten. I don't know that that's been even carefully done.

Has anybody looked at rapamycin administration and anti malarian hormone level for example, once, let's say a woman is already in her AMH decline but hasn't fully bottomed out to zero. Could you rescue some of that? I mean, because that if you look at the physiology of that it is a monotonically decreasing function and it is very steep and if you could simply stop it from declining, that would be remarkable, let alone turn it in the other direction. Again, here's an example of you could study this for hundreds of thousands of dollars. These are nuts, not large sums of money. It would have to be paid for by somebody who's just genuinely obsessed with knowing the answer and not realizing they can't make a buck off this.

Right. We talked about the human studies that are going on now. So the one I'm most familiar with is out of Columbia. So Yushin Su, who directs the Reproductive Aging center at Columbia, and Zev Williams are leading this clinical trial. That's one of the biomarkers that they're looking at. I don't think they have any data yet. And this was a grant that was funded by the Impetus Grants Foundation. So these are smaller grants, but like you said, you can actually start to answer some of these questions and it doesn't require tens or hundreds of millions of dollars to start to gather some data. So they have a clinical trial that is ongoing. I don't know. I've actually got a call with them next week. So I don't know how far along they are. Except I do know that there are some patients in the trial now looking at at women with premature ovarian failure and it's a double blind, placebo controlled randomized clinical trial. With rapamycin. So I think that will start to get more data around safety in a younger, healthier population and hopefully start to get some data around potential efficacy for ovarian function in people. I want to just mention, though, I mean, I'm extremely pleased that the Impetus Grants foundation funded that trial. They're also funding a periodontal disease trial out of the University of Washington. Washington. I'm grateful to them for doing that from, like, a scientific perspective, but I'm also extremely frustrated that the funding for these kinds of trials is so small. And these trials are, let's be honest, they're underpowered for what we would really want to do. If you really want to answer the question, you're not going to get there with trials of 40 people. That's not enough. The researchers are doing the best they can in the system that we're working within. But what happens is you end up with these small clinical trials that give a hint of efficacy and show no problems in terms of safety. But then it takes another two years, another three years, another four years to get a grant to take it to the next stage. And that's why it ends up taking two decades with something like rapamycin to actually get to an answer. Whereas if this was a rapalog, an mtorc, one specific rapalog, you could go out, start a company, and raise money to do a more accelerated path. I don't mean to dismiss that approach, but we have a perfectly good drug here with lots of human safety data that probably works. And it's frustrating, to say the least, that things have gone so slow.

It really is, because people don't appreciate what it takes to go from IND to phase three approval. And the fact that that's already been done for this molecule, and basically all you're really doing is a series of new phase 2 and phase 3 trials on an approved drug is such an enormous tailwind. I share your concern. David. One kind of last thought from you. You're quite close to the landscape of this. You've personally been involved in the development of a number of Rapa logs. What is your take today of the landscape? Landscape in this arena?

It's so funny how the winds change. I would say 10, 12 years ago, if you went and said, I want to target mtor, the universal response was, we have rapamycin. It's off patent, it's cheap. No, thank you. Let's move on. I think now there's almost been a complete reversal, as Matt kind of alluded to. Right. You can come up with small differences in rapamycin, which you can still sort of patent protect and apply them to much more niche applications, and people are potentially willing to fund them. These are not the mega biotechs that are being started, but certainly enough to get things going. And so I think there's a general consensus that MTOR matters as a target. What is frustrating to me, if Matt mentioned his frustrations, sure, I think rapamycin and its derivatives are great and we should do exactly what Matt was saying and somehow incentivize that. I personally think, though, there's actually a whole bunch of other targets in that pathway that may be more beneficial. For example, one thing we didn't get to here, here, and Matt alluded to this, it's very clear that the response to nutrient deprivation is not just mtor at all. In fact, that nutrient sensors, we found, clearly talk to a whole bunch of other processes. And so if you want to get closer to that nutrient deprivation state, one has to go to those. And so right now, the way I read it is people are willing to invest modestly in molecules that are rapamycin derivatives. They still, though, have the mindset mtor is drug. And therefore, if you want to go to other components of the pathway, which I think we don't have time to discuss them, would be more interesting. There's really not an appetite for that.

Gentlemen, I have a list of pages in front of me of topics that I wanted to discuss with you that extend far beyond rapamycin and mtor. We've very, very briefly touched on a couple of them. We've talked a little bit about epigenetics. We've talked a little bit bit tangentially about some of the other hallmarks of aging. We've had hints of other questions that are remarkable, questions that seem so basic and fundamental, and yet it's amazing we don't know the answer to them. Questions such as why do different organisms age at different rates? Why do different organisms of similar size have different lifespans? These are all some of the most interesting questions in biology and questions that we collectively as friends discuss all the time privately. But I think it would be really enjoyable to have one of these discussions publicly in this way. So I think the only thing to say here is we should just probably sit down collectively and do this again much sooner than when we plan to go back to Rapa Nui, which, guys, I have that on the calendar for 2026. Let's find time between now and then to sit down and do a part two of this discussion, which I hope was half as enjoyable for you guys as it was for me.

It was great. Peter. Thank you so much. It's always so much fun to talk to you and Matt to hear all your thoughts. Thank you.

Yeah, anytime guys. This has been a blast. I like the three way podcast here, Peter. It works.

Yeah. Or we could do it in person the next time, guys. It's even more fun in person.

Definitely. Yeah.

And remember, Peter, when we go back to Easter island, we got to bring the plaque.

I know we are bringing the plaque.

We went to the place where the soil sample was collected. There's supposed to be a plaque and the plaque was stolen. So we're going to do that.

Yeah, we will indeed. All right, guys, thank you so much. So much.

Thank you, thank you.

Thank you for listening to this week's episode of the Drive. It's extremely important to me to provide all of this content without relying on paid ads to do this. Our work is made entirely possible by our members and in return we offer exclusive member only content and benefits above and beyond what is available for free. So if you want to take your knowledge of this space to the next level, it's our goal to ensure members get back much more than the price of the subscription. Premium membership includes several benefits. First, comprehensive podcast show notes that detail every topic, paper, person and thing that we discuss in each episode. And the word on the street is Nobody's Show Notes Rival Hours Second, monthly Ask Me Anything or AMA episodes. These episodes are comprised of detailed responses to subscriber questions typically focused on on a single topic and are designed to offer a great deal of clarity and detail on topics of special interest to our members. You'll also get access to the show notes for these episodes, of course. Third, delivery of our Premium newsletter which is put together by our dedicated team of research analysts. This newsletter covers a wide range of topics related to longevity and provides much more detail than our free weekly newsletter. 4 Access to our private podcast feed that provides you with access to every episode including AMAs, sans the spiel you're listening to now and in your regular podcast feed. Fifth the Qualys, an additional member only podcast we put together that serves as a highlight reel featuring the best excerpts from previous episodes of the Drive. This is a great way to catch up on previous episodes without having to go back and listen to each one of them. And finally, other benefits that are added along the way. If you want to learn more and access these member only benefits, you can head over to PeterattiMD.com subscribe you can also find me on YouTube, Instagram and Twitter all with the handle Peteratti MD. You can also leave us review on Apple Podcasts or whatever podcast player you use. This podcast is for general informational purposes only and does not constitute the practice of medicine, nursing or other professional health care services including the giving of medical advice. No doctor patient relationship is formed. The use of this information and the materials linked to this podcast is at the user's own risk. The content on this podcast is not intended to be a substitute for professional medical advice, diagnosis or treatment. Users should not disregard or delay delay in obtaining medical advice from any medical condition they have and they should seek the assistance of their healthcare professionals for any such conditions. Finally, I take all conflicts of interest very seriously. For all of my disclosures and the companies I invest in or Advise, please visit PeterAttiamD.com about where I keep an up to date and active list of all disclosures.

Sam.