272 ‒ Rapamycin: potential longevity benefits, surge in popularity, unanswered questions, and more

1. 0:00 – 1:00

   Intro

   1. MKMatt Kaeberlein0:00

      You know, 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. Um, and I think through all of that, the one thing that, that has kind of 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 healthspan, um, in complex animals. Uh, rapamycin also seems to positively impact pretty much every aspect of healthspan that we measure.

   2. PAPeter Attia0:48

      [upbeat music] Hey, everyone, welcome to The Drive podcast. I'm your host, Peter Attia. Well, guys, we're gonna

2. 1:00 – 12:00

   David & Matt’s expertise in mTOR & rapamycin

   1. PAPeter Attia1:00

      try something a little different today, which is we're gonna try to have a three-way discussion, which, um, is something we would easily be doing, uh, if we were sitting over a meal, but always makes for a slightly more challenging podcast. That said, given, uh, our familiarity with each other and your familiarity with this topic, I am a hundred percent confident this is gonna 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, uh, a little over five years ago. And, uh, Matt, you and I have spoken a number of times since. David, you and I, uh, at least on the podcast, have not, obviously in person all the time. So with all that said, um, many people are gonna be new to this topic. They will have heard a lot about it. They may have even read a chapter about it in my book, which you guys were both very gracious to help, uh, me fact check and edit. But here we are. We're gonna pretend that someone coming into this discussion doesn't really know anything about rapamycin, doesn't really know what this mTOR thing is, and 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. So 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. Um, let's start with you, David. You, you've worked on this molecule, uh, rapamycin, your entire scientific career, going back to your PhD. Um, and here we are, 30-plus years later, you're still the leading authority on it. Can you just tell us a little bit about that?

   2. DSDavid Sabatini3:01

      Sure, Peter. Thank you for having us. Uh, and, and nice to see you, Matt. Um, so no, in- indeed, I've worked on rapamycin my whole life. Uh, when I was a student with Sol Snyder at 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 a protein that we now call mTOR, which, uh, Michael Hall, uh, had identified a yeast version of this that Matt, uh, was one of the early workers on this called TOR1. And since that time, we've done a lot of biochemistry to try to figure out what this protein does. And at the end of the day, what we conclude in a kinda 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, and I think that accounts for why mTOR has so many different roles because if you think about 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, we, we tend to forget that now because of course we're in a, tend to be in an 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, and really the work that I'm the most satisfied with is how it senses nutrients, uh, and the nutrient sensors themselves, which are the, the actual proteins that bind the small molecules that tell, uh, mTORc1 in particular that it detects nutrients. And so I'm excited to, to be here and to, to delve into some of the implications of this work.

   3. PAPeter Attia4:36

      Awesome. Thanks, David. Matt, um, people who are, you know, listeners of this podcast are, are gonna be maybe a bit more familiar with you because in addition to that very first podcast we did circa two thousand eighteen, um, 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, um, maybe for folks who are hearing you for the first time today, can you give kind of a, 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?

   4. MKMatt Kaeberlein5:13

      Sure. Well, um, first of all, thank you for having me back. I'm glad you aren't sick of me yet. And, uh, it's fantastic to be here, uh, with, with David. Um, and I wish I could say that, that I was smart like David was and recognized immediately how important mTOR would be and rapamycin. But I, um, 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. Um, 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, we found that we could also increase 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 Uh, appeared to affect the biological aging process not only in yeast but also across the animal kingdom. Um, and we now know even in, uh, mammals like mice and potentially in larger mammals like dogs and people. So I think with that knowledge, um, you know, 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. Um, and I think through all of that, the one thing that, that has kind of 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 pre-clinical studies, that we know about today for impacting not only longevity, but to the extent that we can measure various metrics of healthspan, um, in complex animals. Uh, rapamycin also seems to positively impact pretty much every aspect of healthspan that we measure. So, so I've continued to study it for that reason. And I think, um, probably what I'm maybe best known for these days is pushing forward a, a veterinary clinical trial of rapamycin to really start to answer the question of, you know, 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, um, am quite passionate about, about, about continuing to push forward and see, see where we end up.

   5. PAPeter Attia8:06

      And I guess I should probably, um, before I make my next comment, d- 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, as a conflict if people want to consider that a conflict. Uh, that, uh... But, but regardless, um, I think what's really great about having you guys together, [clears throat] and tell me if you agree with this assessment, is on the continuum of understanding rapamycin and mTOR, David, you know, 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, i.e. translational research. And [clears throat] obviously the bedside in this case is, is not just the bedside of humans where we aren't quite yet, but really the, the bedside of more complex mammals. So would you guys kind of agree with that assessment that, that your skill sets and your knowledge base and your research are very complementary through that continuum of bed to bedside?

   6. DSDavid Sabatini9:08

      I, I, I would. I, I would add though that, that Matt, you know, 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, that, you know, are, are extreme, I think Matt has been very careful. And, and, and I think 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, and as I've told many people who know me well, I, I pretty much put Matt in one of the most respected categories of aging researchers for that reason. All right. So-

   7. PAPeter Attia9:45

      Yeah. I, I, I agree with that completely.

   8. MKMatt Kaeberlein9:48

      Thank you for that. [laughs]

   9. PAPeter Attia9:48

      [laughs] I, I, I share your, your view.

   10. MKMatt Kaeberlein9:49

       You guys are embarrassing me. Come on now. [laughs]

   11. PAPeter Attia9:52

       [laughs]

   12. MKMatt Kaeberlein9:53

       Uh, Peter, let me just give a little bit of a, a, a twist on what you said. I mean, I agree with, with, with what you said, uh, conceptually. I would also say I think, you know, even though much of my research on mTOR and rapamycin has been what people would typically consider preclinical or basic research, um, it is different. And I, I agree. I think the, the approach that David has taken throughout his career is quite complementary in many ways to, to the approach that I've taken in that, you know, David has really, a- as you already said, been the pioneer and the leader in really understanding detailed mechanistic aspects of the whole mTOR signaling network. Um, 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, that David and, 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, to maybe more applied contexts and clinical applications.

   13. PAPeter Attia11:07

       I think that's a, that's a beautiful way to describe it. And I also want to echo something that David just said, which is I, I, uh... 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 the sort of 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, um, 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, uh, a commercialization route. And the, 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, and I'm, I'm actually, uh... and I think everybody should be very grateful

3. 12:00 – 19:05

   The discovery of rapamycin & first use in humans as an immunosuppressant

   1. PAPeter Attia12:00

      for, 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, and I, I probably even write about it in, um, in the chapter of my book. I, I do think that the discovery of rapamycin is the place to begin this because, you know, there's a very unique Uh, [chuckles] 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, y-you and I got to visit this very special place where the bacteria that ultimately produced this drug was discovered. We, we certainly have plans to go back. It's on our, it's on our list of things to do in the next few years, and we shall. Um, do you wanna give folks the, the story of, of how this, this, this molecule rapamycin came to be?

   2. DSDavid Sabatini12:50

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

   3. PAPeter Attia12:54

      He certainly will. [chuckles]

   4. DSDavid Sabatini12:55

      Anyone who cares about rapamycin would say-

   5. MKMatt Kaeberlein12:56

      Yeah, yeah. For the record, I did try to get this recording done on Rapa Nui, so I just wanna put that out there. [laughs]

   6. PAPeter Attia13:02

      We, we'll be doing-

   7. MKMatt Kaeberlein13:03

      So I'm in

   8. PAPeter Attia13:03

      ... we'll be doing another one there, for sure.

   9. DSDavid Sabatini13:06

      Well, as, as you know, Peter, there, there were in our attempts by pharmaceutical companies to collect soil samples and other biological containing samples throughout the world. And, and there Wyeth-Ayerst did come into possession of a soil sample from Easter Island, uh, otherwise known as Rapa Nui in the, in the South Pacific, at one point claimed to be the most remote island in the world. I think it's, it's actually technically not, but very far from anything. And in that soil sample, actually in, in Canada, people eventually isolated bacteria from it, bacteria called, um, Streptomyces hygroscopicus. 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 in, in 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. Um, and, and like was done at the time, these molecules that these, these bacterial products, what you, you really would call an antibiotic, it did come from bacteria, uh, were tested, you know, in many different assays. And I actually think, and Matt may, may, may correct me, I think some of the earlier assays were actually immunological assays even before some of the, uh, antifungal assays. Uh, and, 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 of rapamycin and some of the targets, uh, were first identified 'cause of the ease of genetics. Um, so this is a story that began, I think the original soil samples may have even been in the '60s.

   10. PAPeter Attia14:38

       Yeah, I think it was '66 or '67-

   11. DSDavid Sabatini14:41

       Yeah

   12. PAPeter Attia14:41

       ... soil samples. And then-

   13. DSDavid Sabatini14:42

       Exactly

   14. PAPeter Attia14:42

       ... Suren I don't think really got around to digging into it until '71 or '72.

   15. DSDavid Sabatini14:48

       Exactly. And, and then he championed it. In fact, you know, one of my most valued possessions that when I started working on rapamycin, we, we, we didn't have much. And, and Sol Snyder, my advisor, wrote Suren and asked for some. He sent us many grams, which I later calculated had a street value of many hundreds of thousands of dollars [laughs] if, if it had, uh, if one could sell it like that, and a, and a really nice note wishing me luck, and the entire bibliography of rapamycin at that time, which consisted of his papers and a couple of abstracts. So it's a, it was a, it was a little thin book at, at the time. And he is the one who championed it, and eventually it took... The, the clinical path took way too long, and I think that even impacted some of its utility because the patents expired I think before, uh, you know, you could 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, right? And I think an, a question that we could ask ourselves, and I think we will, is rapamycin as good as it gets? Right? There obviously are der- 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?

   16. MKMatt Kaeberlein16:01

       So I think, you know, something David said there we may again also touch on, which is the, 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, so I do wonder whether the history of rapamycin and the rapidity at which it, it i- 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, the reputation that the drug got as a dangerous drug, um, based on the way it was developed clinically. So I think that's an important piece of the puzzle here to think about.

   17. PAPeter Attia17:13

       Yeah. Just to give some numbers to it, right? The first paper that Suren Sehgal 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, you know, when you sort of make a, a chemical discovery, file an IND, and work all your way through. Second point I'd make is, uh, you know, as a, as a, as a former surgical resident who... So, so, you know, I was in my surgical residency taking care of transplant patients when rapamycin was in full use. Now, a-again, it's interesting, David, another drug that the whole reason you got involved in rapamycin was because of FK506, which was, uh, uh, another dr- cousin of rapamycin, um, that if, if I recall, the whole reason your lab was using rapa was as a control that didn't have sort of the calcineurin, uh, properties of FK506. But that's sort of a, an interesting footnote. Um, but we were giving rapamycin out constantly. And to your point, Matt, it was a drug that was typically given two to three milligrams a day every single day, but with three other drugs, right? You were also getting prednisone, CellCept, MMF. You were 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, right? Which is for, you know, the better part of a decade, nineteen ninety-nine to two thousand and nine, the only experience that the scientific world has with this is in that context. And yeah, you're gonna see a lot of side effects, but how do you know they're from rapamycin? I mean, and how do you know that

4. 19:05 – 25:53

   Emergence of rapamycin as a molecule with the potential to prolong lifespan

   1. PAPeter Attia19:05

      they would be the same elsewhere? So, so what, what happened in, in two thousand and nine that kind of changed this? And, 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 that first-- in, in the buildup to that first study in two thousand and nine?

   2. DSDavid Sabatini19:39

      You know, I was not very aware of it, I have to say. But I, I do have to say that, um, once we started making that connection of rapamycin to nutrients, right, which, which many groups did, uh, if, if you actually look at the history of it, and, and it was already appreciated for many, many decades before that things like caloric restriction had an impact on lifespan, right? And 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, uh, to have an impact. Um, and so we, we had found no impact. And, and then there were genetics that came out in, in worms and, and, and Matt's work. And so-- and a lot of other people really pioneered the aging space, not us, uh, at all. But I remember when that paper came out, right? I think it was a Nature paper that came out reporting rapamycin as one of the, the bigger hits in, in, in the ITP study. And I think what happened there is it-- and, and I think Matt said this before. It, it connected, you know, his work in yeast and the work in other organisms with a mammal, right? And, and that, that now seems-- We just take that for granted, right? 'Cause as Matt said, it, it, it does impact all those t- those different animals and, and single-cell or- 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, right?

   3. PAPeter Attia21:06

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

   4. DSDavid Sabatini21:25

      I don't know if there, there are, but, but certainly dietary restriction, yes, in one form or another. And that did link-

   5. PAPeter Attia21:32

      Mm-hmm

   6. DSDavid Sabatini21:32

      ... all those organisms and, and as far as I know was, was all done before rapamycin, before the discovery of TOR. So there was this universal intervention. I think even in, in bacteria people have shown impacts on, you know, replicative, uh, 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 a mimetic and potentially having this impact. So, um, so I don't know, uh, Peter, whether there are specific molecules that do that. I would-- I'd probably-

   7. PAPeter Attia22:04

      I'm not aware of any. I mean, yeah, I'm not, I'm not asking the question rhetorically. But I'm-- I agree with you that outside of caloric restriction, um, which by the way doesn't universally extend life. There are models where, and, and certainly times at which that can be administered when it is not-

   8. DSDavid Sabatini22:21

      Right

   9. PAPeter Attia22:21

      ... uh, a life-extending, uh, strategy. But, but yes, I mean, rapamycin in that sense stands alone as the, the-- Unless, Matt, you can think of a counterexample that I'm missing.

   10. MKMatt Kaeberlein22:31

       Yeah. I'm, I'm gonna try to respectfully tell you guys that you're wrong. [laughs]

   11. PAPeter Attia22:35

       Okay.

   12. MKMatt Kaeberlein22:36

       So, so here's what I would say. Rapamycin for a small molecule is probably the only pharmacological intervention that has been, uh, reproducibly shown to robustly increase lifespan and healthspan 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, 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-

   13. PAPeter Attia23:18

       Right

   14. MKMatt Kaeberlein23:18

       ... 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, that we did with Brian Kennedy and Daniel Promislow, it was probably two thousand and seven, 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-

   15. PAPeter Attia23:56

       Mm-hmm

   16. MKMatt Kaeberlein23:56

       ... 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, of longevity. So I just think it's important for me, I guess, to, to, 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, with a lot of this, um, 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 gonna 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, um, 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, um, that's useful to just re-emphasize 'cause a lot of people have lost track of that.

   17. DSDavid Sabatini25:08

       So, so I, I completely agree, Matt. And, and when I just saw Peter in Austin, and he took me on a rocking, uh, trek in 104 [laughs] degree heat, we exactly talked about this topic. And, and my point was that sort of biochemical, cell biological processes that are conserved amongst all these organisms are going to be the ones that are gonna impact aging. And in fact, I tend to dismiss those processes which are, are, are less conserved as potentially r- causing, impacting the aging process. I 100% agree to you that whatever is the fundamental issue that happens in cells that leads to aging is gonna be conserved, and therefore, the regulators of that process or the impactors of that process will be conserved.

5. 25:53 – 36:26

   Groundbreaking rapamycin study on mouse lifespan extension & the open questions about the timing & frequency of dosing

   1. PAPeter Attia25:53

      I wanna come back to this point because I s- I, yeah, we had an amazing... If we had been able to record that ruck session, if you could eliminate all the huffing and puffing-

   2. DSDavid Sabatini26:03

      I have a brain

   3. PAPeter Attia26:03

      ... uh, it would've been a great podcast in and of itself.

   4. DSDavid Sabatini26:06

      Mm-hmm.

   5. PAPeter Attia26:06

      And we're gonna come back and talk about some of those things. This is actually a great step off to make a point that what we're talking about here is the broad term of geroprotection. And I always kinda 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 healthspan 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. Um, 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, right? 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, right? Very few organisms have ApoB, i.e., very few organisms succumb to ASCVD. So, you know, that doesn't mean that we shouldn't look at disease specific tools to modulate lifespan and healthspan, but what we're talking about here is so much more fundamental. And, um, 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 eight of them together.

   6. MKMatt Kaeberlein27:41

      We can get seven, I think.

   7. PAPeter Attia27:41

      Yeah, I don't know.

   8. MKMatt Kaeberlein27:41

      We could do this like Name That Tune, right?

   9. PAPeter Attia27:43

      Yeah, yeah.

   10. MKMatt Kaeberlein27:43

       How many hallmarks of aging can you... [laughs]

   11. PAPeter Attia27:45

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

   12. MKMatt Kaeberlein27:48

       There's, there's actually 12 now, right, with the new and improved version.

   13. PAPeter Attia27:51

       Oh, there is? My God, I'm, I'm so dated. Um, okay. So let's now, um, dive into mTOR a little bit, unless... Were you gonna say something else?

   14. MKMatt Kaeberlein28:04

       Yeah, can we go back to that mouse study? Yeah, just, just for a minute. So we-

   15. PAPeter Attia28:06

       Oh, go back to the ITP. Yeah, sure.

   16. MKMatt Kaeberlein28:08

       Yeah, 'cause there's another part of that study. So just, 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, 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 a, 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, uh, rapamycin, uh, uh, 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 chow 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.

   17. PAPeter Attia29:19

       Yeah.

   18. MKMatt Kaeberlein29:20

       So, so it was a happy accident. But in my view, I, I, 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 sort of opened up what we now consider to be routine, which is that you can actually have an impact on longevity and some, at least some metrics of healthspan when you start treatment in middle age. And as we have started as a field to think about translational application, that becomes hugely important, 'cause 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, from the perspective of actually being able to implement.

   19. PAPeter Attia30:02

       Especially when you consider what David said at the outset, right, which is mTOR is the master regulator of how nutrients trickle into the system. Are you gonna be in an anabolic state or are you gonna be in a catabolic state? Well, you know, 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, and yes, you, your, your, your telling of that story is remarkable, and I think also speaks to the serendipity that often lies, uh, in, in scientific discovery. It's often an accident or something going wrong that leads to that. Because you're right, I don't think... And I've talked about this, I think, with Rich Miller. Nobody would've ever... They, they were contemplating sacking the whole study because-

   20. MKMatt Kaeberlein31:03

       That's right

   21. PAPeter Attia31:03

       ... they couldn't get the rapamycin formulated.

   22. DSDavid Sabatini31:06

       It is a fascinating question though

   23. MKMatt Kaeberlein31:08

       why that when the starting point of delivery of rapamycin does have an impact on the life extension and the healthspan extension. And what, what the, what the biological basis of that-

   24. PAPeter Attia31:19

       Mm

   25. MKMatt Kaeberlein31:19

       ... is something that at least I don't have a great conceptualization of that. And, and, and, and I'm sure Matt has thought about this much more, but, but it, it's interesting to think about how one, one designs experiments to try to ask that question.

   26. PAPeter Attia31:33

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

   27. MKMatt Kaeberlein31:40

       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-

   28. PAPeter Attia31:51

       Correct

   29. MKMatt Kaeberlein31:51

       ... as the sort of primary metric-

   30. PAPeter Attia31:53

       Mm-hmm
6. 36:26 – 47:13

   Explaining mTOR & the biology behind rapamycin’s effects

   1. PAPeter Attia36:26

      Yeah. So, so, so David, let's now start to talk about the how. So, um, I think it's worth doing this in a little bit of detail. Um, and I know that this might, the next few minutes might be among the most technically, um, perhaps challenging for, for, 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, that move on. And, and I think it's also important to understand how nutrients work, right? So we're gonna talk a lot about amino acids and probably in particular leucine. Um, so in, in any order that you feel it makes sense to, to walk us through that, David, explain-

   2. MKMatt Kaeberlein37:19

      Yes

   3. PAPeter Attia37:19

      ... how this molecule, mTOR, which sits at the epicenter of our existence as living entities on this planet. How does it do its job?

   4. DSDavid Sabatini37:35

      Sure, happy to talk about this. You know, and I think the one thing to, for the listeners to understand is that rapamycin is, is quite unique in, in another aspect that we haven't talked about, but also was very exciting at the time, right? Rapamycin, unlike most drugs, right, 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. And, and as, as people like Stuart Schreiber have pioneered, it's, it's really a molecular glue that connects mTOR and FKBP, and that interaction is absolutely critical. Um, so how does, uh, uh, mTOR work? So, you know, when, when we first and others found mTOR, it was this big protein. Uh, it looked like a kinase. That is, it's a protein that puts phosphates onto other proteins. Uh, but yet what it did, what its targets were, were completely unclear. And I think as we were talking in the pre-session and Matt pointed out, it's incredibly complicated. It, it probably acts on hundreds of other proteins. Now, 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, and on the breaking down side, as you and I, Peter, have discussed, and I'm sure Matt agrees, it's au- autophagy, right? The, 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, on the catabolic, uh, side. And the way mTOR works... And, and you know, 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, a moribund-

   5. PAPeter Attia39:43

      Mm-hmm

   6. DSDavid Sabatini39:43

      ... protein. And, and the critical breakthrough was the idea at some point that mTOR must work by being bound to other proteins. Now again, this seems, like, obvious, right? Like, everyone talks about the TOR complex. But, 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. But what we came to realize, and this goes back to sort of serendipity, it turns out the detergents, right? So when, when you have a mammalian cell that's surrounded by a lipid, sort of a 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.

   7. PAPeter Attia40:33

      Yeah.

   8. DSDavid Sabatini40:33

      Why does it? We don't know. And when we moved to other detergents, used things to stabilize it, we then found these TOR complexes, right? And, and the first breakthrough for us was the discovery of a protein that, that got this name raptor, um, which, uh, at the time people didn't like this name but now is, is a well-studied protein. And as Matt alluded, there's actually genetics on raptor that connect it to, to lifespan and the aging process. And so that defined what we now call TORC1 or MTORC1. Another protein that we named Richter defines what we call MTORC2. I'm sure we'll talk about, uh, 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. Like, 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, in sort of a biological lingo we call upstream, right? 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. And, uh, 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, an organelle called the lysosome. Um, the, 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. So at, at that, uh, 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, uh, afterwards.

   9. PAPeter Attia42:37

      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, things like that.

   10. DSDavid Sabatini42:55

       Yeah, in terms of-- That's a good question, Peter. In terms of numbers, w- we're talking certainly thousands of, of complexes in existence. It's not, it's not an amazingly rare protein, right? 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. Um, but not, their proteins are much, much less abundant than that. Um, and it's distributed actually quite evenly between MTORC1 and two, at least in the cells that, that we have looked, uh, in, uh, in, in culture. When you look across tissues in, in a mouse or a rat, it's actually pretty even across tissues as, as, as well. And so to some extent that puts it in the What sometimes pejoratively is called a housekeeping protein, right?

   11. PAPeter Attia43:41

       [laughs] That's gonna be uninteresting, right?

   12. DSDavid Sabatini43:44

       Now, those turn out to be some of the most important. Exactly. Some of the most important proteins in the cell. So what, what we have found now, and I think others would agree, is that regulation of mTOR levels itself is, is, 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, we have to start thinking about also for new modalities to, uh, to target mTOR. Um, you know, we know a lot more now.

   13. PAPeter Attia44:15

       So we'll, 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, you know, 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?

   14. DSDavid Sabatini44:45

       There actually is some in RBCs.

   15. PAPeter Attia44:47

       Interesting.

   16. DSDavid Sabatini44:47

       Which has been very confounding to us 'cause RBCs don't have things like lysosomes in them. They-

   17. PAPeter Attia44:51

       Yeah. There's so much that's been missing

   18. DSDavid Sabatini44:52

       ... there's even some, there's even some in platelets. Um.

   19. PAPeter Attia44:54

       Or mitochondria for that matter.

   20. DSDavid Sabatini44:55

       I, I've actually always wanted to go and look in RBCs for this, uh, for this reason. As far as we can tell, every cell has some mTOR and MTORC1.

   21. PAPeter Attia45:03

       Hmm.

   22. DSDavid Sabatini45:03

       And it... And I would argue, and, and I'm not sure if I'm 100% correct in this, I would argue that in almost every cell, MTORC1 is a very critical protein for the health of that cell, right? And, and Matt alluded to a study, I guess, where some, where people have used now catalytic inhibitors, right? And, 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, right? 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. Uh, it's, so, so it doesn't fully inhibit all the activities of even MTORC1.

   23. PAPeter Attia45:51

       So let's give people an analogy, David. So if the, if the place where... So for example, in this case, if the amino acid is like a baseball that's supposed to bind in the, right 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 shape, it changes the shape of the glove so that the intended target doesn't. Is that a good analogy?

   24. DSDavid Sabatini46:20

       It is. Now, now, the thing that binds in the glove here is ATP, which is the phosphate donor, and then the substrate, let's say S6 kinase.

   25. PAPeter Attia46:26

       Got it.

   26. DSDavid Sabatini46:27

       But you're, you're exactly right, right? Those things are... 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 to that cave, but you haven't fully blocked that entrance.

   27. PAPeter Attia46:43

       Mm-hmm.

   28. DSDavid Sabatini46:43

       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, like as you alluded to, shape changes and stuff. But the simplest way to think about it, it's a steric block of st- some things, but not others.

   29. PAPeter Attia46:58

       Perhaps also worth just re-mentioning that this is the MTORC1 cave, right?

   30. DSDavid Sabatini47:03

       Exactly.
7. 47:13 – 51:20

   Differences in how rapamycin inhibits mTOR complex 1 (MTORC1) versus mTOR complex 2 (MTORC2)

   1. DSDavid Sabatini47:13

      and, and Matt, you said that there's been a study now on lifespan or, or, or, 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, I've not seen this. Uh, but you're right. The catalytic inhibitors basically annihilate the activity of MTORC1 and MTORC2 if used at the right dose. Rapamycin partially inhibits MTORC1 and over time can also partially inhibit MTORC2. So they're very dramatically different h- how they act.

   2. PAPeter Attia47:45

      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 1 that will eventually but not immediately lead to the inhibition of mTOR complex 2?

   3. DSDavid Sabatini48:08

      Sure. So, so, uh, before I'll, I'll say that MTORC1, its canonical substrate is S6 kinase. So every biologist looks at S6 kinase phosphorylation as an indicator of MTORC1 activity. The canonical substrate for MTORC2 is a protein called AKT. Everyone looks at AKT phosphorylation as a canonical output. And so I had this postdoc, one of the more con- colorful people I had, a guy from Kazakhstan, actually, Dos Sarbasov, who had discovered Richter and the AKT phosphorylation. One day he comes in my office. He's like, "David, rapamycin inhibits MTORC2." And I was like, "Dos, that is impossible," because we had tried to show that this FKBP rapamycin would bind to MTORC2 and it wouldn't bind. It would bind fine to MTORC1, but it wouldn't bind to MTORC2. 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. But over in this-

   4. PAPeter Attia49:10

      What year is this, David?

   5. DSDavid Sabatini49:12

      Wow. Um, this would have been, you know, early 2000s and somewhere in that range, right? I'd, I'd have to look back. May- may- maybe 2003, 2004, in that range.

   6. PAPeter Attia49:24

      Yeah.

   7. DSDavid Sabatini49:24

      I think we published the paper maybe in 2005.

   8. PAPeter Attia49:27

      Okay.

   9. DSDavid Sabatini49:28

      Um, 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 has, and convinces you of what turns out to be a, a pr- pretty important discovery, but I didn't believe. And so, and so the reason was why did this happen, right? Because you could take MTORC2, you could put FKBP rapamycin on it, and MTORC2 would phosphorylate AKT no problem. It didn't care. Totally fine. You do the same experiment with MTORC1 and S6 kinase, and now you could really inhibit, uh, S6 kinase phosphorylation. So what we came to realize though, and to some extent it's, it's obvious, is that of course MTORC2 is not born as MTORC2. It's born as mTOR and raptor, and they have to find each other, right? 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 raptor can't bind. So you can't make MTORC2. And so what happens then, Peter, is that when you incubate a cell in a mouse over prolonged periods of time of rapamycin, you basically, all your mTORs acquire an FKBP rapamycin, and therefore you can't form a raptor complex, right? And so the way that we're preventing MTORC2 formation, and therefore MTORC2 inhibition, is drama- 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 raptor, to come together. Basically, FKBP rapamycin is preventing that interaction. And the way people are getting around this, which I don't think we're gonna discuss, is by, is by understanding that at a better biophysical level, which we now do have that understanding.

8. 51:20 – 56:42

   Reconciling the biochemical mechanism of rapamycin with its longevity benefit

   1. PAPeter Attia51:20

      So M- 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?

   2. MKMatt Kaeberlein51:40

      No, it doesn't surprise me, but I think, I think the reason it doesn't surprise me is in, in part, I think we need to, again, recognize that this network is extremely complicated. So the model that, 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, to really understand in a, in a detailed way. So part of the reason why I'm not surprised is be- is [chuckles] is because we kind of already knew all the longevity outcomes before we understood this biochemical mechanism, right? 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 healthspan 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 healthspan 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, that the benefits of rapamycin come from MTORC1 inhibition i- 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.

   3. DSDavid Sabatini53:13

      Mm-hmm.

   4. MKMatt Kaeberlein53:14

      But that's incomplete. So I guess it's all to say that I think, and, and this is, you know, 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, and I'm not convinced at this point that the idea that, 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, how accurate that model is. It's a model that still needs to be studied.

   5. DSDavid Sabatini53:52

      So Peter, can, can I add something? I-- So I completely agree with Matt. I, I think that last statement is 100% true. I, I don't-- I think we almost have no evidence, uh, that, to, to make that decision one way or another. But I think the reason if MTORC2 is i- 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 s- the amounts of, of rapamycin used in the longevity studies are relatively modest. Um, they probably still are somewhat intermittent even though a mouse is eating them, right? Because of course it doesn't eat all the time. Unlike what we were doing experimentally where we were dosing rapamycin very high, keeping it above, you know, a certain level, and certainly in tissue culture it's twenty-four/seven. And you can imagine that once an mTOR finds a raptor, it's immune to rapamycin now. So, so as soon as one of those guys interact, you're gonna have an MTORC2, and you need very little MTORC2 to keep AKT happy. We, we, we found that early on. You only need probably ten to fifteen percent, at least in cells in culture, to keep AKT happy. So there's gonna be escapers. As soon as rapamycin goes besor- below a certain amount, there'll be escapers and you'll make an MTORC2. So I, I, I do think we have to ask how relevant that activity is to the potentially beneficial effects of rapamycin. And, and, you know, and a lot of the drive to find rapamycins that don't do that comes from my work, right? And so to some extent, um, I'm saying, "Hey, look, i- is that oversold?" I think that is a potential, you know, argument to make. And, and Matt alluded to, there, there is-- I would almost argue there's no perfect experiment to answer this question because mTOR is shared. You almost can't answer this. That's almo- almost a philosophical issue.

   6. MKMatt Kaeberlein55:40

      And one thing I wanna add, 'cause I think this is, David kind of said this, but I think it's really important for people to appreciate, 'cause sometimes we get into the, 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, right? And so what David said about you don't need a lot of MTORC2 activity to survive and, and the same thing's probably true for MTORC1, but rapamycin is kind of turning down MTORC1 immediately a lot, and that's gonna depend on the dose of rapamycin that we give. And then over time, turning down the MTORC2 knob, 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, 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. Yeah.

   7. DSDavid Sabatini56:35

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

   8. MKMatt Kaeberlein56:40

      Yeah, yeah.

   9. DSDavid Sabatini56:41

      And so...

9. 56:42 – 1:04:43

   Important discoveries about the interplay of amino acids (leucine in particular) & mTOR

   1. PAPeter Attia56:42

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

   2. DSDavid Sabatini57:05

      Sure. So, so this also has a little bit interesting backstory. So when I first identified mTOR in Sol Snyder's lab, I talked to my dad, who is a cell biologist. And, uh, and he said, "David, you have to localize mTOR within the cell." [lips smack] And I kind of... To be honest with you, I kinda dismissed that in maybe a little bit 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 in 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?" Right? And I didn't get any definitive answer. Then the rabbit died. The antibody was lost. And literally for about, that would've been in '93 or something, literally until probably 10, 15 years later, we did not revisit this question. And 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, as I mentioned. But then he did-

   3. PAPeter Attia58:13

      Sorry, just to make sure, just to make sure we understand, David.

   4. DSDavid Sabatini58:15

      Yeah.

   5. PAPeter Attia58:15

      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-

   6. DSDavid Sabatini58:21

      Yes

   7. PAPeter Attia58:22

      ... "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.

   8. DSDavid Sabatini58:29

      Yeah, I think, you know, people didn't... They saw dots inside the cell. Now it was clear that there was a little vesicles and I, and I think I, I probably, you know, if I had sat down... This was, like, literally walking around the hallway, right?

   9. PAPeter Attia58:40

      [laughs]

   10. DSDavid Sabatini58:40

       So maybe if I had sat down with more experts and, and, and really showed them more experiments that we, we would've 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, and, and there was, I would say, no good antibody to do this experiment for the next 15 years. And this guy Tim got one, [lips smack] and he showed that, again, we saw the same punctate pattern that I had seen as a student, you know, 15 years earlier. Um, but he then went on to figure out what it was, and they were these things called lysosomes. Again, these sort of recycling centers. They're, they're... 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, 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 sort of 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, and we could go into the details of this, but it's, it's probably a little bit, uh, 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, you know, it 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 sort of protein real estate used to do this, which tells you the cell cares about this. So the question becomes which amino acids? And, and I have to say that, that really, that, that was broken open not by us, but by Joe Avruch. He had a paper in JV- JBC where he looked at amino acid regulation of mTOR. This was, this is before the lysosomes. He was looking at the activity of using S6 kinase. And he basically found a couple amino acids that matter. He found leucine, you know, a, a, uh, very common essential branched-chain amino acid, an important component of, of whey protein, for example, that people take. Arginine, uh, a very basic amino acid, technically not essential. Lots of nitrogen in, in that amino acid. And those were the two big ones that, that he found. Now since then, we have found others. And, 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, um, you know, used in farms, that leucine does cool stuff like boost satiety, you know, a feeling of, of having fed, uh, boost muscle mass. And eventually we found it. We found the receptor for leucine. It's a protein called sestrin. And, and, and for me, you know, you have in your scientific career, I think you only have a couple of moments where you're kinda moved 'cause 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, uh, in sestrin where you're like, "Okay, like, this is how nature does it," right? So from eating a steak to now detecting the leucine in that steak, there it is. It's nestled in there. And then you can sort of imagine how it, uh, goes on to talk to mTOR. Um, and so, um-

   11. PAPeter Attia1:02:49

       This was-

   12. DSDavid Sabatini1:02:49

       So for us that's been-

   13. PAPeter Attia1:02:50

       This was, was this Bobby that led this work?

   14. DSDavid Sabatini1:02:53

       So yeah, so, so Rachel, uh, Wolfson and Lynn Chantranupong, they had discovered sestrin as the sensor for leucine, and they could show that genetically, biochemically. And then Bobby Saxton t- working with us and Thomas Schwartz, he then did the crystal structure-

   15. PAPeter Attia1:03:08

       Mm

   16. DSDavid Sabatini1:03:08

       ... of, uh, of, of leucine bound. And, and what was beautiful about that structure was it immediately said it's gotta be leucine, which we had kinda, we and others had shown already, right? You could, you could try isoleucine, but it didn't work. Um, and so you could see it nestled in there and you could see all the parts of sestrin 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, right? The, the immediate idea was, "Hey, can we mimic the anabolic effects of leucine without taking leucine? Can we make something better than leucine?" And, and we've managed to make things slightly better, but nothing dramatically better. And the structure tells you why, 'cause it's, it, it basically is made to fit leucine and nothing else.

   17. PAPeter Attia1:03:54

       How long does leucine stay in that pocket?

   18. DSDavid Sabatini1:03:58

       You know, 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.

   19. PAPeter Attia1:04:14

       Hmm.

   20. DSDavid Sabatini1:04:15

       And that there actually may be an active way of getting leucine out. That lid has some very interesting sort of 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, but I think you hit upon something that suggests that this is not so, you know, it's not that leucine is popping in and out. It pops in, but probably getting out requires an active step.

10. 1:04:43 – 1:12:00

    Reconciling rapamycin-mediated mTOR inhibition with mTOR's significance in building & maintaining muscle

    1. PAPeter Attia1:04:44

       So Matt, what does -- what, how, 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 healthspan. 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, i.e. anabolic activation, is good. And yet we've just made a very compelling case for why blocking that extends lifespan. How, how would you start to reconcile what seems conflicting?

    2. MKMatt Kaeberlein1:05:59

       Yeah. So I mean, um, it's, i-i-it's obviously ex- going to be extremely complicated. I think I'd start by going back to, to a point that I made five minutes ago or so, which is that these are not on/off switches, right?

    3. PAPeter Attia1:06:12

       Mm.

    4. MKMatt Kaeberlein1:06:12

       So you really need to think about this in the context of what is the optimal level of mTOR complex 1 activity for whatever, whatever it is that the cell, the tissue, the organ, the organism needs to do to function or stay alive, right? So certainly we know that you need mTOR activation to build new muscle. Um, 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 would-- That was the prediction that was made. The reality turns out to be the opposite. Uh, 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 in, during aging. Um, that's a different question though than I think, which is one that a lot of people ask, which is if, if, 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, don't think we have that data. And 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. My intuition is-

    5. PAPeter Attia1:08:17

       Which is a shame because it's been done with metformin, right? Like, there's no reason we shouldn't know that question.

    6. MKMatt Kaeberlein1:08:22

       Uh, sort of. So sort of. So metformin, you're, you're talking about in, in the human studies or-

    7. PAPeter Attia1:08:26

       Yeah, in the humans

    8. MKMatt Kaeberlein1:08:27

       ... or in rodents?

    9. PAPeter Attia1:08:27

       No, in the humans.

    10. MKMatt Kaeberlein1:08:27

        Yeah. So people have looked a little bit, a little bit, although again, I would say even there the data's 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, uh, cardiovascular-

    11. PAPeter Attia1:08:43

        Cardio, yeah

    12. MKMatt Kaeberlein1:08:43

        ... training. Yeah. And I, I'd say the data is unclear, although there is some reason to think that metformin might impair the, what people think of as the positive responses to exercise. Complete tangent-

    13. PAPeter Attia1:08:54

        Yeah

    14. MKMatt Kaeberlein1:08:54

        ... but I agree with you. The fact that that has- hasn't been done for rapamycin in humans is a shame, and it should be, and hopefully will get done sometime in the, in the near future. Um, but to get it back to your question, I mean, I, I, I wish I could tell you why that's the case. I'm just sort of telling you what the observations 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, uh, 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-

    15. PAPeter Attia1:09:31

        Mm-hmm

    16. MKMatt Kaeberlein1:09:31

        ... 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, you know, detailed mechanistic answer to your question because people haven't really started to disentangle, um, those things. The last, 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 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 be, uh, have an impact on, on sarcopenia in, 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-

    17. PAPeter Attia1:10:44

        Mm

    18. MKMatt Kaeberlein1:10:44

        ... effects on longevity. But because mice are not, 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 gonna have those same beneficial effects in people where sarcopenia seems to be much, I would, I would argue at least, much more important for quality of life, probably life expectancy, but certainly quality of life in, in older adults. So I just want to make that caveat that we, we need to be a little bit careful about extrapolating from mouse studies to, to humans in the context of muscle preservation, muscle function, and sarcopenia.

    19. PAPeter Attia1:11:20

        Yeah. I, I think that's actually really important, and, and 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, right? If you're living in a sterile environment where there aren't curbs to step off and places to fall and injure yourself, um, I mean, one only need look at the mortality data for people over the age of 75, even over the age of 65 if they fall. Um, it's, it's an enormous cause of not just death, but morbidity. T- total-

11. 1:12:00 – 1:18:09

    Unanswered questions around the tissue specificity of rapamycin

    1. MKMatt Kaeberlein1:12:00

       Yeah

    2. PAPeter Attia1:12:00

       ... destruction of quality of life. Uh, I want to ask you both a question, or you can both chime in and whoever's feels more com- more, uh, more [laughs] whoever has a stronger point of view on this. Do we know... Maybe I'll start with you, David. Do we know from the laboratory in, uh, in, in, in, 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, uh, in the, in the adipose tissue? I mean, uh, 'cause, 'cause this would factor into it, right? There's also, uh, uh... You know, in a, 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, uh, complex 1 specificity. So, so, uh, David, anything you, you can add on that?

    3. DSDavid Sabatini1:12:57

       Sure. So, so, you know, I think, you know, Matt answered your question perfectly well, and I think it shows you the complexity of the issue, right? It's not only MTORC1 versus MTORC2, it's which cell type, right?

    4. PAPeter Attia1:13:07

       Mm-hmm.

    5. DSDavid Sabatini1:13:07

       Is it, is it muscle fibers? Is it in, in inflammatory cells, immune cells? At what dose? Is it which process? Is it 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. You typically need to do a little bit of a loading dose, but you'll get it into the brain. Now, there's been some discrepancies. Some people say immediately. In our hands, it usually took a, a couple of doses to get in the brain. At the lower doses, though, that are used in-

    6. PAPeter Attia1:13:40

       Sorry, and was, was that, was that a couple of doses without interruption?

    7. DSDavid Sabatini1:13:45

       Yeah. D- d- typically where we did not let a tro- a trough level to get too low, right? So this, this would've been probably in a mouse maybe every eight hours or something, maybe every 12 hours. So it's pretty aggressive type of dosing.

    8. PAPeter Attia1:13:57

       Okay. 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?

    9. DSDavid Sabatini1:14:08

       Probably, with, with classic rapamycin. And, and, and, and there was some, you know, in terms of the pharma world, right, people that wanted to treat tuberous sclerosis where you get these sort of tumors in the brain. They did not think rapamycin was adequate for that because of CNS penetration. But, but again, very talented people have argued differently-

    10. MKMatt Kaeberlein1:14:27

        Sure

    11. DSDavid Sabatini1:14:27

        ... uh, than that. But, but our experience certainly was that the brain seemed more resistant. In fact, sometimes you would get, you would stain the brain, you'd see almost like a peripheral inhibition, like it kind of had permeated a little bit from sort of-

    12. MKMatt Kaeberlein1:14:39

        Hmm

    13. DSDavid Sabatini1:14:39

        ... you know, blood vessels in the, in the sort of, uh, dura and stuff. But, but now, but, but I think the more relevant question, Peter, is at these lower doses that people take potentially for, for healthspan, lifespan studies in the ITP studies, what i- what are the tissues that are most affected? Matt may know, but, but, but I don't know. And my feeling is that it's not gonna be so equal in those situations. Those are very low doses compared to what we would give to rapamycin. And so my, my, my bet is that there's much more variation, and that might actually be very interesting to, to know. You 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-

    14. MKMatt Kaeberlein1:15:30

        Hmm

    15. DSDavid Sabatini1:15:30

        ... in the brain, which one has the most prolonged lifespan, healthspan impact? I don't- Matt, I don't think that study's been done, right?

    16. MKMatt Kaeberlein1:15:37

        Not systematically. There's a little bit of data on hypomorphic, uh, mTOR alleles. Um, and gosh, I wish I could remember-

    17. DSDavid Sabatini1:15:45

        They're not tissue specific though

    18. MKMatt Kaeberlein1:15:46

        ... the outcomes of these. Hmm? Yeah, no, there's been a little bit. Veronica Galvin's done some stuff for dementia brain aging. Um-

    19. DSDavid Sabatini1:15:53

        Okay

    20. MKMatt Kaeberlein1:15:53

        ... I don't know about lifespan. I think Torren Finkel may have done something, but I, I, but, but in general, it has not been done outside of, you know, if it's been done, there was like a, a adipose specific knockdown, knockout. I, and, uh, maybe liver specific, but certainly not systematically looking across different tissues.

    21. DSDavid Sabatini1:16:13

        Right. A- and you know, and this does get at this question is like, okay, there's lifespan, but then there's also the health of all the different-

    22. MKMatt Kaeberlein1:16:18

        Yeah

    23. DSDavid Sabatini1:16:18

        ... tissues. And, and my bet would be that you actually wanna impact all tissue. You know, I used to go to aging meetings-

    24. MKMatt Kaeberlein1:16:24

        Hmm

    25. DSDavid Sabatini1:16:24

        ... and I would always challenge when at the speaker dinner I would say, "Tell me a biological system that does not age." Right? Give me one where you don't see the impact on aging. Give it from the biochemical cell biological to physiological level. And as far as I know, no one has ever told me one. So, um, 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, um, and even, you know, Matt alluded to this before, everyone looks at S6 kinase and its substrate S6, phospho S6. Does that m- You know, there's not that much evidence it matters so much for lifespan. There's some. What are the real relevant targets there?

    26. MKMatt Kaeberlein1:17:07

        Yeah. So let me just add on 'cause I think a lot of what you said, David, is, is spot on and may be worth extending a little bit. So this last point about which substrates, um, 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 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

12. 1:18:09 – 1:26:40

    Rapamycin’s ability to cross the blood-brain barrier & its potential impacts on brain health & neurodegeneration

    1. MKMatt Kaeberlein1:18:09

       the real answer is we don't know. I think this brain, 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, 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, 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-

    2. DSDavid Sabatini1:18:57

       Hmm

    3. MKMatt Kaeberlein1:18:57

       ... in older animals compared to younger animals.

    4. DSDavid Sabatini1:19:00

       Hmm.

    5. MKMatt Kaeberlein1:19:00

       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, um, in aged animals and, and in aged people potentially. But I don't know of any real data to support that. So it's, these are all questions that, that I think need answers, and there just isn't much out there right now.

    6. PAPeter Attia1:19:24

       Couple of questions, um, and then, and then a, a, a follow-up comment. Um, what's the size of rapamycin? How, how physically large is it?

    7. DSDavid Sabatini1:19:33

       It's almost exactly 1,000 Daltons, right? It's right, right below 1,000. So, so it, in the world of small molecules, it's a big small molecule, right? A lot of small molecules are more... And so 1,000, uh, the, the, a hydrogen atom is eight Dalton.

    8. PAPeter Attia1:19:45

       Mm-hmm.

    9. DSDavid Sabatini1:19:46

       Right? So it's about 1,000 hydrogen atoms sort of weight. Most small molecule is more in the 2 to 3 to 400 range. So this is, this is big.

    10. PAPeter Attia1:19:54

        And what's the, what's the size at which you can easily traverse the blood-brain barrier?

    11. DSDavid Sabatini1:20:00

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

    12. PAPeter Attia1:20:05

        I see. So it's more about, it's, it's more about the solubility than it is-

    13. DSDavid Sabatini1:20:08

        Yeah

    14. PAPeter Attia1:20:09

        ... yeah, than size.

    15. DSDavid Sabatini1:20:09

        I, I think, um, you, you almost sort of need to I, I almost see like a lot of it get tr- trapped in the membrane. You almost sort of need to sort of push it, uh-

    16. MKMatt Kaeberlein1:20:17

        Mm-hmm

    17. DSDavid Sabatini1:20:17

        ... through. And you know, and the brain has a lot of things like myelin, which are all very lipophy- So I think the, the, I think there's almost like a s- the way I always saw it is a bit of a sink of sort of trapping rapamycin in places that maybe it's not so effective.

    18. MKMatt Kaeberlein1:20:28

        Hmm.

    19. PAPeter Attia1:20:29

        The only anecdote I would add here, and I, I don't know if it means anything, and I, 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, uh, in, in humans of course, to look for, um, 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, you know, 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 cerebrospinal fluid. But not only does that come with the case of, you know, a lot of radiation and potential morbidity respectively for those procedures, it's simply not practical if you're, you know, uh, 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 again, this is very anecdotal, but for our very high-risk patients who are showing amyloid already in the plasma, um, I believe we have put two of them on intermittent rapamycin, so anywhere from five to eight milligrams once a week. And in both cases, the C2N score has improved, meaning, uh, you know, every three months when we are checking the amyloid concentration, it's going down. Now, there are 10 leaps of faith you'd have to take there. Does that mean the amyloid is going down in the CNS as w- I mean, you know, I, I, I don't have to spell that out to anybody who's reasonably thoughtful. Um, but, you know, I think your point, David, about by definition these, these patients are aging, so maybe their CNS, their blood-brain barrier is not as robust, and maybe that even though that's a very low and clearly, uh, 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 and-

    20. MKMatt Kaeberlein1:22:41

        Let me stop you there because I actually want to present a different hypothesis-

    21. PAPeter Attia1:22:44

        Okay

    22. MKMatt Kaeberlein1:22:44

        ... which is that it's actually the periphery that may matter for the brain. And there's two lines of evidence-

    23. PAPeter Attia1:22:49

        Very interesting. Yeah

    24. MKMatt Kaeberlein1:22:49

        ... that I can point to that, that, that, that might support this. Um, one is, you know, we've worked for many years in, in my lab in a mouse model of a childhood mitochondrial disease called Leigh syndrome.

    25. PAPeter Attia1:22:59

        Mm-hmm.

    26. MKMatt Kaeberlein1:22:59

        It's a complex one deficiency in the mitochondria, but it's a brain disease, so it's an, 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 one. 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-

    27. PAPeter Attia1:23:34

        Hmm

    28. MKMatt Kaeberlein1:23:34

        ... tissue signaling piece, and that could be metabolic. You could imagine inhibiting mTOR complex one 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. Not at all, I'm not at all saying the mechanism there is the same as-

    29. PAPeter Attia1:23:50

        Sure, sure

    30. MKMatt Kaeberlein1:23:50

        ... 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, uh, 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- 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 there's, you know, it's plausible that the, that this is at least partly right. Could easily imagine rapamycin's effects on the peripheral immune system would, 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 in, um, at least in laboratory animals.
13. 1:26:40 – 1:40:41

    Rapamycin may act as an immune modulator in addition to immunosuppressive effects

    1. PAPeter Attia1:26:40

       being done. Uh, this gets to a broader point. Um, we've already alluded to the incredibly low, uh, pardon me, slow, uh, timeline for rapamycin's transition into humans, and the net result of that was a drug that, you know, 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, so everolimus, uh, which I believe at the time was sort of part of the Novartis portfolio. Is that correct?

    2. DSDavid Sabatini1:27:18

       I think so, yeah.

    3. PAPeter Attia1:27:19

       Um, so tell us how, how does, how does everolimus differ from rapamycin before we get into talking about one of the more important studies in humans?

    4. DSDavid Sabatini1:27:28

       I don't quite remember, but it's a s- it's a small modification. I thought it was a methyl group on-

    5. PAPeter Attia1:27:32

       Wow, okay

    6. DSDavid Sabatini1:27:32

       ... on rapamycin. You know, the, the, ironically, the original patents on rapamycin did a very poor job of covering obvious derivatives of rapamycin. I w- I was involved in some intellectual property cases that basically boiled down to, uh, in the patents, they talk about rapamycins. And, and Wyeth, who, who owned those patents, Wyeth Ayerst, that, 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, that almost many chemists would come up with, and everolimus is one of those.

    7. PAPeter Attia1:28:12

       Yeah, and-

    8. DSDavid Sabatini1:28:12

       Since then, there's been more sophisticated variations, but everolimus is, I think, Matt, a simple, a simple variant, right?

    9. MKMatt Kaeberlein1:28:18

       I, I think you're right. And again, maybe just for, for context, correct me if I'm wrong, David, but I think we can say that, you know, 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 1. And I think the real differences are more around bioavailability, maybe tissue distribution, and, uh, uh, 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 gonna get differences in peak and trough levels based on the, the bioavailability and clearance, and then maybe some differences in tissue distribution. But, but I think those are the primary things that, that differentiate the rapalogs. Biochemically, I think they're all pretty similar.

    10. DSDavid Sabatini1:29:11

        Yeah. No, I, I, I tend to view them, and certainly in cells and culture, they act identically. I think from a biochemical point of view, they're, they're-- At least the original rapalogs were pretty much identical. And in many cases, frankly, and 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, uh, than rapamycin. I think, I think even Wyeth Ayerst did that, right? They had a molecule, I think CCI-779, I think was its name, which was a simple rapamycin derivative. And, 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.

    11. PAPeter Attia1:29:50

        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 l- we, we, we alluded to something that happened that was really remarkable, if I'm not mistaken. I think it was even April of two thousand nine. I, I kinda remember this pretty well. Um, fast-forward five years, [clears throat] five and a half years, I suppose. It's December of twenty twenty-four. Um, I didn't kn- yet know you guys. You and I wouldn't meet, David, for another year. But, um, 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 geroprotective agent. Because despite 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 God, if, 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 Mannick, Lloyd Klickstein, and others that seemed to at least challenge the very foundation of that. And of course, Matt, you already made a lot of, uh, good points about this, which is, you know, that thinking about rapamycin might have been a bit premature. So, uh, either one of you guys, why don't, why don't you walk us through the, the study in the, uh, the Australian senior citizens that I think for many people was, um, I don't know, for me at least, a, a huge turning point in how we thought about this, this drug.

    12. DSDavid Sabatini1:31:43

        I- And all I'll say to me is that that paper, the Joan Mannick paper with rejuvenation of the immune system, I think will be seen in the aging field as certainly a milestone paper along with the ITP paper. 'Cause as, 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, was mind-blowing. Um, I think Matt can speak more-

    13. MKMatt Kaeberlein1:32:07

        Yeah

    14. DSDavid Sabatini1:32:07

        ... to the, the details of it and-

    15. MKMatt Kaeberlein1:32:09

        I, I would agree completely with that, and, and we may wanna come back and touch on that because I think, uh, as people are thinking about clinical trial endpoints for gerotherapeutics, 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 gerotherapeutic. 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 Zheng's lab in mice that preceded the, the Joan Mannick paper where they showed that you could treat with rapamycin for, I think, six weeks in that study, um, and rejuvenate the immune function of a mouse. And to me, the one experiment in there that is most compelling is they, 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, um, either a, uh, 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. And then 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, uh, demonstration of immune rejuvenation in an aged mammal. So I think that study is what really set the stage and allowed Joan and, 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 laid out, except of course they didn't give people lethal doses of influenza. But what they did do was they, they enrolled healthy older people, I think they were over the age of 65, and they, they couldn't have... There were some set of preexisting disease that they, that were, they would be excluded for. So they were considered relatively healthy for their age. Um, and they got either placebo or they t- 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 the in- there are a few interesting things here. So they got, I think, everolimus for six weeks or a placebo, and they got either five milligrams-

    16. PAPeter Attia1:35:18

        I think it was... Yeah, it was five milligrams once a week, 20 milligrams once a week, and I think it was one milligram daily was the third group, was beyond the placebo.

    17. MKMatt Kaeberlein1:35:25

        That was what I was going to say. So I think between the two of us we're, we're close, if not spot on.

    18. PAPeter Attia1:35:28

        Yeah.

    19. MKMatt Kaeberlein1:35:29

        Yeah. So, so there are some interesting... And then they, and then they gave... So that was for six weeks, and then they gave a flu vaccine, and then they looked at antibody titers and, and, and I think, I don't know if it was this study or a later one where they looked at, um, 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 five milligrams once a week and one 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, um, 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-

    20. PAPeter Attia1:36:31

        Yes, 320 if I'm not-

    21. MKMatt Kaeberlein1:36:32

        ... hundreds of people.

    22. PAPeter Attia1:36:32

        Yeah, I think it was about 80 per arm.

    23. MKMatt Kaeberlein1:36:35

        Yeah. 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 five mgs once a week group, was essentially no different than placebo. And so I think it, 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, you know, may- and this I think is getting w- what you were talking about, Peter. Maybe it is possible that lower doses of a rapalog 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, kind of independent of the potential immune rejuvenating effects, which I don't want to minimize 'cause that's hugely important. I actually think both of these things are, are important things that that study kind of set the stage for.

    24. PAPeter Attia1:37:34

        And I think from that study comes a word, I don't know if it was, you know, certainly it wasn't coined in that study, but, but in my mind at least, it went from, uh, we shouldn't think of this as an immune suppressant. We should think of it as an immune modulator.

    25. MKMatt Kaeberlein1:37:49

        Right.

    26. PAPeter Attia1:37:49

        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, the 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, these are not just T cells, but you know, this is part of the, the cellular immune system. So, so that also I think is a very interesting footnote to this story.

    27. MKMatt Kaeberlein1:38:25

        I was living through this at Hopkins, you know, the, the, the age of the immunosuppressants. I mean, remember how miraculous cyclosporine seemed?

    28. PAPeter Attia1:38:32

        Yeah.

    29. MKMatt Kaeberlein1:38:32

        And then FK506. And rapamycin to some extent got caught up

    30. DSDavid Sabatini1:38:36

        In being this generic sort of immunosuppressant. But the truth is, when you looked at the data in cells and culture, in, uh... It's actually not so easy to inhibit in some of those sort of 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, as you and, and Matt have said, that has sort of persisted, but it never kinda looked
14. 1:40:41 – 1:49:00

    Might rapamycin induce changes in T cell methylation patterns, potentially reversing biological aging?

    1. PAPeter Attia1:40:41

       Let me ask you guys a question. We're gonna 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 Mannick, Klickstein study, do you believe that you would see a change in the methylation pattern pre and post rapamycin?

    2. MKMatt Kaeberlein1:41:04

       Absolutely. But, but I think what you're really asking is would we, would we see a change in the methylation pattern that is what people are calling a reversal of biological aging. I don't know if that's what you're asking, but-

    3. PAPeter Attia1:41:16

       It is. It i- it's exac-

    4. MKMatt Kaeberlein1:41:17

       So there's no question in my mind you [laughs]

    5. PAPeter Attia1:41:17

       It's exactly where I'm going, which is-

    6. MKMatt Kaeberlein1:41:19

       Yeah

    7. PAPeter Attia1:41:19

       ... given our shared interest in that topic as well-

    8. MKMatt Kaeberlein1:41:21

       Yeah

    9. PAPeter Attia1:41:21

       ... which is, you know, when we-- 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?

    10. MKMatt Kaeberlein1:41:39

        Yeah. I don't have a strong feeling ab- I don't, I don't have a strong enough feeling to make a, a strong prediction there. Uh, like I said, there's no question you will see a change in, in the epigenome, but that's kind of just saying everything is-- Everything big that you do to a cell is gonna affect the epigenome. I'm less convinced that these epigenetic clocks are really measuring what, from a, from a biological aging perspective, what some people think they're measuring. And so I'm, I'm-- 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. What, what-- First of all, what are the canonical age-related epigenetic changes in T cells, and how closely are those linked to the, the functional declines that we see with T cells that go along with, with aging? I don't think that's really been carefully fleshed out. And so, 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's gonna reverse it.

    11. PAPeter Attia1:42:45

        No, I, I think the current versions of the clocks are not measuring anything that's of interest, truthfully. But I-- But, but I still wonder if we just don't have the technology yet to, to actually read this at CPG resolution, and therefore-

    12. MKMatt Kaeberlein1:42:59

        Yeah

    13. PAPeter Attia1:42:59

        ... we don't really know what the heck is going on, right? When we use these crappy microarrays to read these things, when we're sort of averaging out methylation patterns, I, you know... Look, I, I think it's like trying to play the piano with mittens on. It's totally unhelpful. Um, but if you can take the mittens off and put your fingers on, it's a different sport.

    14. DSDavid Sabatini1:43:20

        You know, to get to Matt's point, we had actually tried to look at the impact of rapamycin on specific methylation, uh, patterns, not only on, on the DNA itself, but also on histones and, and using a variety of different tools. And the truth is, we found almost-- And we never published this 'cause we almost found nothing specific, and all the impacts really were from the cell cycle delay.

    15. PAPeter Attia1:43:41

        Hmm.

    16. DSDavid Sabatini1:43:42

        Once you sort of normalize that away, you couldn't say, "Hey, mTOR inhibition's regulating K twenty-seven 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 gonna f- s- see impacts.

    17. PAPeter Attia1:43:57

        But, but why, David? So, so, so that's very interesting. But how would that explain what we just saw, that in six weeks, which is nothing in the s- in the s- in the span of a person's lifetime, six weeks of inhibiting mTOR... And again, let's do it in the mouse experiment 'cause that's so much more dramatic, right? And now admittedly, six weeks might be analogous to, you know, 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. How, y- how-- 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.

    18. DSDavid Sabatini1:44:48

        But I mean, Peter, this gets to the fundamental question here is what is wronged, wrong with the aged lymphocytes, and what does rapamycin do to them to fix that, right? And, and so what I'm telling you is that cells in culture... Like, we always imagine there's a signal transduction pathway from MTORC1 to a specific epigenetic change.

    19. MKMatt Kaeberlein1:45:06

        Mm-hmm.

    20. DSDavid Sabatini1:45:06

        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, right? Like a cell state. You know, Rick Young always used to say, "Epigenetics is the, is the setting of the state, not the thing that gave you that state at the beginning," right? And this is an important distinction.

    21. MKMatt Kaeberlein1:45:35

        Correct.

    22. DSDavid Sabatini1:45:35

        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, 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 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.

    23. MKMatt Kaeberlein1:46:07

        And let, let me just add a, a couple of, couple of thoughts here. So one is, you know, uh, if you think about, go back to the hallmarks of aging, which, you know, there used to be nine, now there's 12, epigenetic changes is only one of the hallmarks of aging. And you can, you can find evidence in the literature that rapamycin impacts all 12 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, but I, I think again there's reason to think this is at least conceptually partly the case. We know that with aging there's... It's not that immune function declines globally, right? 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, that one of the things rapamycin is potently doing is knocking down this hyperactivation. And this is something I've wanted to mention but, but, but we didn't talk about. In both the Manick study and the Pan Jiang 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 se- receive rapamycin-

    24. DSDavid Sabatini1:47:49

        Mm-hmm

    25. MKMatt Kaeberlein1:47:49

        ... 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 r- 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, um, to account for the observation that 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.

    26. PAPeter Attia1:48:55

        Which begs a question, um, to cycle or not to cycle, right?

15. 1:49:00 – 1:59:32

    Rapamycin side effects & impacts on mental health: fascinating results of Matt’s survey on off-label rapamycin use

    1. PAPeter Attia1:49:00

       So, so Matt-

    2. DSDavid Sabatini1:49:01

       Right

    3. PAPeter Attia1:49:01

       ... you, you, you wrote, um, or co-authored a paper that came out earlier this year that was a, a survey, not an experiment, but a survey that looked at over 300 users of rapamycin. So this is a, you know, a bunch of people who are clearly using rapamycin off-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, uh, nearly 200 if I recall, who were, you know, hopefully as similar as possible in terms of their health consciousness, which would be an obvious confounder, uh, but who were not rapamycin users. Can you give us some of the, the highlights of what that survey discovered?

    4. MKMatt Kaeberlein1:49:42

       Right. So, 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, with a study like that, um, because it was all self-reported, all survey based. We got, in some ways, lucky in the sense that, 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 that, the, the responses that the individuals gave to the surveys... I don't have it sitting in front of me, but, um, you know, 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 did, uh, had a swath of just Middle America. But for what it, for what it's worth, they seem to be pretty similar. And so, um, there were a few take homes from that, from that study. I think the, the biggest take home for me is that there really was no evidence when you look between the people who were using rapamycin off-label and the people who'd never used rapamycins for significant side effects of, of any sense other than mouth sores. That was in fact the only... We had one of the surveys was a list of, I think, 30 or 40, you know, 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, uh, have you experienced any of these in the past three months? And then for people who'd never used rapamycin, same question. The only thing that came out as statistically significantly more common in the rapamycin users was mouth sores, and that makes perfect sense. That's the most commoning, uh, side effect that, that organ transplant patients experience, and lots and lots of people who've used... I think, Peter, you've talked about-

    5. PAPeter Attia1:51:30

       I, I, I have a wicked one-

    6. MKMatt Kaeberlein1:51:31

       ... your experience with mouth sores, right?

    7. PAPeter Attia1:51:32

       I have a wicked one at the base of my tongue-

    8. MKMatt Kaeberlein1:51:34

       Yeah

    9. PAPeter Attia1:51:34

       ... right now that I almost burnt before this podcast. Um, and it's my-

    10. MKMatt Kaeberlein1:51:40

        So, so in a sense, that's a nice-

    11. PAPeter Attia1:51:40

        ... it's my only biomarker

    12. MKMatt Kaeberlein1:51:41

        ... positive control.

    13. PAPeter Attia1:51:42

        I was just about to say-

    14. MKMatt Kaeberlein1:51:42

        Yeah

    15. PAPeter Attia1:51:42

        ... it's my only biomarker-

    16. MKMatt Kaeberlein1:51:44

        Yeah

    17. PAPeter Attia1:51:44

        ... that I know that I'm getting-

    18. MKMatt Kaeberlein1:51:46

        Yeah

    19. PAPeter Attia1:51:46

        ... high-quality rapamycin.

    20. MKMatt Kaeberlein1:51:48

        [laughs] Right. So in a sense, it's, it's nice to see that, and it's interesting-

    21. PAPeter Attia1:51:53

        What's the, what's the approximate-

    22. MKMatt Kaeberlein1:51:54

        ... that was the only thing

    23. PAPeter Attia1:51:54

        ... uh, frequency? Because I think in the Mannick study it was surprisingly low at five milligrams weekly.

    24. MKMatt Kaeberlein1:52:00

        I think it was like-

    25. PAPeter Attia1:52:00

        It was like 15%.

    26. MKMatt Kaeberlein1:52:03

        Yeah, I think it was, like, 15% in ours as well.

    27. PAPeter Attia1:52:06

        Okay.

    28. MKMatt Kaeberlein1:52:06

        Yeah. I think that's exactly what it was, actually.

    29. PAPeter Attia1:52:08

        Okay.

    30. MKMatt Kaeberlein1:52:09

        Um, so yeah. So, so 15-ish percent of people reported mouth sores.
16. 1:59:32 – 2:05:20

    Impact of taking rapamycin in people who contracted COVID-19: more insights from Matt’s survey

    1. MKMatt Kaeberlein1:59:32

       Um, so I think what I would-

    2. PAPeter Attia1:59:32

       Let's go back to the survey. Yeah, let's go back to the survey.

    3. MKMatt Kaeberlein1:59:35

       Yeah.

    4. PAPeter Attia1:59:35

       The other things, I, 'cause I think if I-- 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-

    5. MKMatt Kaeberlein1:59:43

       Right

    6. PAPeter Attia1:59:43

       ... and that's why they're taking rapa 'cause they're, 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.

    7. MKMatt Kaeberlein1:59:56

       Yeah. So let me tell you what we obs- what we observed in the data, right? And again, with all the caveats that, that there are around the way the study was, was designed and, and carried out. So within, there 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-

    8. PAPeter Attia2:00:35

       Throughout

    9. MKMatt Kaeberlein2:00:36

       ... 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 didn't-- there's no reason to believe based on our data that rapamycin impacted the likelihood that somebody would get a positive COVID-19 result. And you know, again, this is self-reported, so we asked people if they, to confirm that this was a, a positive result from a test, but we're going by what they told us, right? So we can't, 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 length of symptoms and hospitalization for each of those groups; and then self-reported long COVID, as in experiencing ongoing symptoms of, of COVID after like a three-month period. Um, 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 we, I don't wanna make too much of it, but signif- 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, how at a, at a crude level we kind of think COV- long COVID in particular is working and s- severe COVID infections, which is there's this hyperinflammatory or chronic inflammatory response. It kinda makes sense that rapamycin use may have benefits in the context of that prolonged in- inflammation or hyperinflammatory response. So that might explain, uh, what we saw in the data. But again, I think it's just suggestive and, 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 is, this may be a general property of rapamycin for a bunch of different types of at least viral infections.

    10. PAPeter Attia2:03:25

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

    11. DSDavid Sabatini2:03:48

        Yeah, no, I, I always used to joke that when I was purifying mTOR I got a huge dosing, and given that-

    12. PAPeter Attia2:03:52

        [laughs]

    13. DSDavid Sabatini2:03:53

        ... uh, given that early exposure is better, I got the benefit then. I, I never wore gloves and, you know, it, it, it's a powder. I remember it would get into my nose and stuff, so I've snorted rapamycin-

    14. PAPeter Attia2:04:03

        Fair point [laughs]

    15. DSDavid Sabatini2:04:03

        ... in- inadvertently. So, so I did get a dose at the time. To, to-- Now, you know, Peter, it, it isn't, it isn't such a willful thing. It's, it's more that, you know, it takes some effort to go and, and, 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 mimetic to some extent of a healthy diet, let's-- 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 that is-- that would be my biggest question. It, it wouldn't be am I afraid of it. I don't, I don't, I'm not. Um, but will it actually do anything?

    16. PAPeter Attia2:04:44

        But, but isn't there sort of a, a per- you know, sort of a hedging or a Pascal's wager, which is if, i-i- as long as you're-- 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 'cause it ain't cheap?

    17. DSDavid Sabatini2:04:59

        Sure. So, so, so I agree, but then that's where the laziness factor comes in-

    18. PAPeter Attia2:05:03

        [laughs]

    19. DSDavid Sabatini2:05:03

        ... and sort of figuring out how to do it and stuff. But, 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, um, Peter, you have, this cyclical nature, right? I'm much more interested in sort of a, a very-- 'cause, 'cause what can't I do, right? So if, if

17. 2:05:20 – 2:09:50

    What David would like to study with mTOR inhibitors

    1. DSDavid Sabatini2:05:20

       I starve myself, what happens? My body synthesizes certain nutrients, breakdowns 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, right? So I can't, through dietary interventions, starve a cell of nutrients like I can, can in a dish. I can't.

    2. PAPeter Attia2:05:42

       Mm-hmm.

    3. DSDavid Sabatini2:05:42

       The body fights that. And of course, eventually you run out of stores and you, and you die, but in a, in a, in a normal type of sort 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. I-- 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 rapamycin.

    4. PAPeter Attia2:06:15

       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?

    5. DSDavid Sabatini2:06:22

       Sure. So the, so the allosteric inhibitor, rapamycin and, and derivatives is, is going to do this partial inhibition of MTORC1 and chronically-

    6. PAPeter Attia2:06:28

       The, the rock that partially obstructs the cave entrance.

    7. DSDavid Sabatini2:06:31

       Exactly.

    8. PAPeter Attia2:06:32

       Yeah.

    9. DSDavid Sabatini2:06:32

       The partial block. And, and also partially inhibit MTORC2, and there's gonna be perhaps some tissue specificity, some kinetic, uh, differences. Um, 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, you know, if we, we have misdosed by, by mistake catalytic inhibitors in a mouse, and a mouse will drop dead.

    10. PAPeter Attia2:07:07

        It-- When you say drop dead, are you talking about the same way where, uh, mitochondrial inhibitors like cyanide, which immediately cease respiration, will kill an animal within seconds? Is it-

    11. DSDavid Sabatini2:07:17

        No, i-it'll take usually a couple hours. The mouse will stop moving. It'll get cold. Sometimes it'll have seizures.

    12. PAPeter Attia2:07:22

        Wow.

    13. DSDavid Sabatini2:07:23

        Um, but, but it, it will die.

    14. PAPeter Attia2:07:24

        But still profoundly and acutely toxic.

    15. DSDavid Sabatini2:07:28

        Profoundly bad, yes.

    16. PAPeter Attia2:07:29

        Yeah.

    17. DSDavid Sabatini2:07:29

        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 anticancer agents. We, we made some of the first ones and, and also were 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-

    18. PAPeter Attia2:08:05

        Mm-hmm

    19. DSDavid Sabatini2:08:05

        ... type of s- study and, and, and potential use because I feel that, again, with diet you can get close to, uh, to rapamycin's, uh, impact. And, and again, this is my, my personal belief w-with some, some data to support it. But what I know you can't get close to with diet is what a catalytic inhibitor can do.

    20. PAPeter Attia2:08:27

        Yeah.

    21. MKMatt Kaeberlein2:08:28

        Can I add a couple things o-onto that? I think, I think you said that in a-- I've, I've tried to make this point before, and I think you, you said it in a way that I've never-

    22. DSDavid Sabatini2:08:35

        Mm

    23. MKMatt Kaeberlein2:08:36

        ... thought about it, or at least I've never said out loud, which is, which is im-important, that, um, you know, rapamycin... I think the, the, the point is that rapamycin is very different than dietary restriction. They're overlapping, but they have lots of differences. And I, 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. I think 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, the equation when people are thinking about diet, uh, and comparing it to, to rapamycin. The catalytic inhibitors, though, the, 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, that hit other, other kinases. David's shaking his head, so you can tell me why I'm wrong. But, but there are other m- other proteins that- That some of these molecules that inhibit mTOR

18. 2:09:50 – 2:20:10

    Joan Mannick’s studies of RTB101 & other ATP-competitive inhibitors of mTOR

    1. MKMatt Kaeberlein2:09:50

       will also inhibit. And RTB101, which we didn't talk about the future, the subsequent studies from Joan at Novartis and then when, 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. And I, I don't know if we know, in terms of the side effect profile, how much of that is due to mTOR, 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 Restore Bio, they did dose people with RTB101 and did not see significant side effects. So they... You know, you can ask whether they saw significant efficacy. That trial actually was shut down. But, uh, 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.

    2. PAPeter Attia2:10:54

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

    3. MKMatt Kaeberlein2:11:05

       They did, with everolimus. So they had two arms.

    4. PAPeter Attia2:11:07

       Okay.

    5. MKMatt Kaeberlein2:11:08

       One was the combination, and one was RTB101 alone. Yeah.

    6. PAPeter Attia2:11:11

       Yeah.

    7. DSDavid Sabatini2:11:12

       Yeah, no. So, so, um, I, I think Matt... Look, I'm sorry. I, I meant to... My, my shaking was that I was agreeing with you.

    8. MKMatt Kaeberlein2:11:18

       [laughs] Okay.

    9. DSDavid Sabatini2:11:19

       And, and, and I was... I actually was... That, that study that, that, that Mannick did after, I was confounded by that study and perplexed because this RTP which they, 101, which they renamed. I think it was MVP103, which was a Novartis molecule that's a dual mTOR PI3K 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's really the po- one of the pioneers in mTOR biology, which is exquisitely specific. The, the, the, the... You can dial out PI3K 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. And they managed... So the molecule we made, TORN1, we never managed to dial out DNA-PK. He did. So this Wyeth compound is a beautiful molecule. When Pfizer bought f- uh, Wyeth, they de-emphasized it in favor of dual-activity inhibitors, which again, I did not agree with. But, so I, 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, uh, for an animal when, when, when you use-

    10. MKMatt Kaeberlein2:12:42

        Yeah. I... And I think, you know, something I would... This gets back to low-hanging fruit that, that hasn't been studied. I would love to see somebody take a panel of all of the NO and 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 am confident... Well, maybe I shouldn't say... I, I'm confident that at least in worms, you will find things that work better than rapamycin 'cause we've already done it. [laughs] 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've studied the most? And that seems like a completely unknown to me at this point.

    11. PAPeter Attia2:13:31

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

    12. MKMatt Kaeberlein2:13:42

        Yeah. Absolutely.

    13. DSDavid Sabatini2:13:43

        Yeah.

    14. MKMatt Kaeberlein2:13:43

        That would definitely be my guess.

    15. DSDavid Sabatini2:13:45

        It, it would be my guess too, but, but, you know, the balance between full MTORC1 inhibition, total MTORC2 inhibition, I don't, I don't, I don't know the answer to that. And, 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, in a mouse. Very hard to dose. You gotta put them in detergents and all these, 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.

    16. PAPeter Attia2:14:22

        Mm-hmm.

    17. DSDavid Sabatini2:14:22

        That's the way that I would want to, to, to do that, to sort of mimic a really strong inhibition of this system and then release and see what happens. Um...

    18. PAPeter Attia2:14:35

        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 PI3K inhibitor. I mean, I-

    19. DSDavid Sabatini2:14:45

        And a dirty one.

    20. PAPeter Attia2:14:46

        E- exactly. Like, I n- 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, help me understand that. 'Cause you wrote, if I recall, Matt, you wrote an editorial on this, if I'm remembering correctly.

    21. MKMatt Kaeberlein2:15:11

        Right. So there were actually three studies. So this, the, the, the study where RTB101 was used alone was actually the, the third, and that was their pivotal clinical trial. There was a second Phase 2 in between the 2014 paper and the, the pivotal where they used a combination of everolimus with RTB101. And I, you know, I wasn't, 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, you know, at least somewhat plausible biological rationale for, 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, the thought was-

    22. PAPeter Attia2:16:06

        Yeah, that's right

    23. MKMatt Kaeberlein2:16:06

        ... if you can both boost vaccine response and enhance, uh, re-resistance to subsequent infections, that might be a combination that was, that was useful. So in the second Phase 2, the RTB101 showed a signal. The RTB101 plus everolimus also showed a signal, but RTB101 alone showed a signal. So the decision was made to go to the pivotal with RTB101 alone. And I don't, I don't know the rationale for that. You could speculate it might have something to do with patent life, right? [laughs] And IP around, you know, longer patent life on RTB101, clearer path to market. I, I don't know for sure, but that's what happened. So there was no everolimus in the, the pivotal Phase 3. There are, there are a couple of things about that trial that are worth just mentioning. Um, so 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, 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. Um, uh, 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 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 3. 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 RTB101 showed a significantly lower likelihood of a future laboratory-confirmed viral infection. So, you know, 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. Um, which is interesting because this wasn't a rapamycin, right? 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, have an impact on immune function in, in the population where it was tested.

    24. DSDavid Sabatini2:18:59

        But it, but it was a very dirty catalytic inhibitor, right? It, it, it impacts multiple PI3 kinases, so.

    25. MKMatt Kaeberlein2:19:06

        Yeah. Absolutely, yeah. 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? Yeah, we don't, we don't really, we don't really know 'cause it is dirty.

    26. DSDavid Sabatini2:19:20

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

    27. MKMatt Kaeberlein2:19:42

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

    28. PAPeter Attia2:19:54

        I don't think that's a skeptical point of view. I mean, [laughs] that, that would be my Occam's razor answer to that question, uh, for sure.

    29. DSDavid Sabatini2:20:02

        But there are now so many rapamycin derivatives, right? That you could... I still imagine you could have picked one up to, to do. But who knows?

19. 2:20:10 – 2:28:24

    Impact of mTOR inhibition on autophagy & inflammation & a discussion of biomarkers

    1. DSDavid Sabatini2:20:10

       You'd have to, I guess, go through a lot of preclinical studies and things, so.

    2. PAPeter Attia2:20:12

       So David, you've talked a lot about the, the, the sort of the impact of mTOR inhibition. You've already talked about autophagy. We've talked about, uh, a reduction of inflammation. We haven't talked a lot about the tamping down of senescent cells and, uh, potentially the reduction of the, their, their s- soluble or secretory factors. Um, we have an impact on, uh, uh, proteomics. I mean, lots of things are impacted. Y- you tend to think, if I'm not mistaken, that the auto- the impact on autophagy is the one that might be most responsible for the kind of life property, the altering benefits we see of that. Do you want to expand on that a little bit? And, and, and Matt, I'm kind of curious to hear your point of view on that as well. What do, what do you think, which pathways, uh, plural, d- 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, right? A topic that the three of us have endlessly, endlessly talked about, right? Which is if we believe this is dominated by autophagy, then we need biomarkers for autophagy. If we believe this is, this is dominated by inflammation, then we need better biomarkers for inflammation. So, so with that said, I'd like to hear your thoughts.

    3. DSDavid Sabatini2:21:33

       Sure. Sure. So, so I... You know, when you think of things downstream of mTOR, right? You, you can do a PubMed search and find mTOR and rapamycin literally connected to anything you want, right? And why is that, right? Either there's a specific signaling pathway to that process, or there's a simpler explanation, which to me is that mTOR is a reg- major regulator of protein synthesis, and if you inhibit mTOR enough, particularly with 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. And, and I, 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 cell, of, 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 TFAP, right? So, uh, so one that I think the-- If you had to put in the molecular target of mTOR that's emerged in the last ten, fifteen years as very interesting and prominent, it'd be TFAP. It's a transcription factor that what it does is promote the production of these lysosomes, these, 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 about, and I do, where they actually tried to look at that, right? They, they did mTOR inhibition, and then they looked at downstream pathways genetically and found the biggest impact of perturbing, uh, autophagy. Part of it is, uh, based on common sense. You know, it, it, 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, right? You, you can't make-- You can't prevent the aging of an old house or much less rejuvenate an old house by having a plumber, having an electrician, right? 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, we almost can't ask the question, what is important downstream of mTOR? Because the answer is that mTOR is special because it does a lot of things, and therefore, we can't find one thing that replicates mTOR. Otherwise, we would've already found those things, right? 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, 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. So sorry, long-winded answer, but-

    4. PAPeter Attia2:25:02

       No. It's, it's very, it's very helpful

    5. DSDavid Sabatini2:25:04

       ... that's my, uh, my view on it.

    6. PAPeter Attia2:25:04

       Um, what about you, Matt? Where do you, where do you end up on this question?

    7. MKMatt Kaeberlein2:25:09

       Yeah, I don't disagree with anything that, that David said, and I think the house analogy is-- it's a-- 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, um, autophagy being an important, maybe a, the most important single downstream directly regulated, uh, mTOR process for a bunch of different, uh, aging, broadly speaking, aging effects, is not inconsistent with the idea that in a mammal or in a person, the anti-inflammatory ef-effects account for many, maybe most of the functional benefits that we see when we treat an old organism, old animal, right? 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, uh, different pathologies. So, you know, for example, uh, in hypertrophy, the effects of mTOR on cell size may be most important. Um, in cancers, the effects of mTOR on the cell cycle may be most important. Those are tied into autophagy.

    8. PAPeter Attia2:26:22

       Mm-hmm.

    9. MKMatt Kaeberlein2:26:23

       So I, I, I don't know that it-- 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 effects on global mRNA translation. So again, that may fit with the idea that context is, is important here for which of these downstream processes are weighted, uh, uh, in a relative sense, most importantly for the effects that we see in the c- 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, in the last five to ten 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, in the context of an aged animal. That seems to me, too, that a lot of the, the benefits that we see in terms of organ and tissue function can be plausibly traced back to that effect of rapamycin.

    10. PAPeter Attia2:27:36

        Very interesting. I mean, I think that would lead us to think that, boy, if we really wanted to get a b- a better handle on dosing, we would, we would wanna look deeper into biomarkers of inflammation. And we do have a-- We have more that we can look at there. I mean-

    11. MKMatt Kaeberlein2:27:50

        Yeah

    12. PAPeter Attia2:27:50

        ... 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 don't have-- We, we've got a whole lot of nothing. By God, it's probably been three years now since I had a, a really interesting discussion with Eileen White about this, who's one of the world's experts on this. And, um, yeah, it was, uh, I don't think I got any argument out of, out of Eileen that we really need, uh, a biomarker here because outside of the lab when you can, you know, afford to take tissue, um, we don't, we don't have

20. 2:28:24 – 2:36:20

    The Dog Aging Project: what we’ve learned from testing rapamycin in dogs

    1. PAPeter Attia2:28:24

       much going on. I wanna pivot for a sec and talk about, um, 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 pivot- 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, than mice are. But they're also not genetically inbred the way mice are. They live in our environment, not a sterile environment. Uh, they consume, you know, food that probably looks a little bit more, uh, like the food we would consume, at least in some cases. Um, so, so, so tell us about what you've learned in this study, which has really occupied, uh, more than a decade of, of your, of your research.

    2. MKMatt Kaeberlein2:29:14

       Right. Yeah, so there's two other things I would add about companion animals and, and dogs in particular, where my, most of my work has been, but, but this is also true for, for cats. Um, one is they age more rapidly than people do, right? So that's super important because th- that means we can actually measure outcomes of interest in the timeframe that's compatible with a, with a clinical trial. Um, secondly, they matter, right? M- 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 healthspan and, and longevity of companion animals from, from that perspective.

    3. PAPeter Attia2:29:53

       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-

    4. MKMatt Kaeberlein2:30:02

       Right

    5. PAPeter Attia2:30:02

       ... nobody actually cares how long mice live or how long C. elegans lives.

    6. MKMatt Kaeberlein2:30:06

       Right. Ex- that's exactly right.

    7. PAPeter Attia2:30:07

       Yeah.

    8. MKMatt Kaeberlein2:30:07

       And, and I would also say it's ridiculous to think we're gonna learn nothing about the-

    9. PAPeter Attia2:30:11

       Yeah

    10. MKMatt Kaeberlein2:30:11

        ... biology of aging in humans from studying companion animals. But yes, even if we say that, there's still value in, in doing these kinds of studies and improving the quality and quantity of life for, for pets. So I've been involved, as you know, for a while now with a project called The Dog Aging Project, which Daniel Promislow, Kate Creevy, and myself, um, started, depending on how you wanna do the math, somewhere between 7 and 12 years ago. Um, with the idea, you know, sort of around what, 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 healthspan 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, increase lifespan, improve multiple healthspan 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 kinda work out dosing, um, and then started the larger clinical trial triad a few years ago, um, 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. Um, we're looking at multiple measures of healthspan, 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, uh, length of treatment, we are powered to detect a 9% change in lifespan, which is towards the lower end-

    11. PAPeter Attia2:32:16

        And, and sorry, Matt, is that remaining lifespan or total lifespan?

    12. MKMatt Kaeberlein2:32:21

        That's total lifespan.

    13. PAPeter Attia2:32:22

        Okay.

    14. MKMatt Kaeberlein2:32:22

        So life expectancy, it's a, it's a bigger number for remaining life expectancy.

    15. PAPeter Attia2:32:26

        Okay.

    16. MKMatt Kaeberlein2:32:26

        Yeah.

    17. PAPeter Attia2:32:26

        Yeah.

    18. MKMatt Kaeberlein2:32:26

        So, and the reason why we, 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, a group of donors who, 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. W- 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, um, uh, cohort size. Couple of other things that are maybe worth just mentioning is that the dogs have to be at least seven years old at the time of randomization, and they can't be sick. They can't have any significant preexisting age-related disease. And, 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 preexisting 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, so that's, that's the study. Um, dogs are still actively being enrolled in-

    19. PAPeter Attia2:33:51

        Any size limitations, Matt?

    20. MKMatt Kaeberlein2:33:54

        Yeah, sorry. 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 aging than a smaller weight, body size population.

    21. DSDavid Sabatini2:34:12

        One quick thing. You know, you always ask me if I take rapamycin, and I, and, and my friends ask me whether they should take rapamycin 'cause they know you and that you take rapamycin, and I always say, "Well, when Matt Kaeberlein's, uh, dog study reads out, if it's positive, I'll take rapamycin." [laughs] So-

    22. PAPeter Attia2:34:27

        It's funny you say that, David. W- I say that to a lot of our patients as well. I say, "Look, I, again, I have A relatively strong conviction. It's modestly held. Um, 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, when we'll have the readout of this study. So yeah, I think-

    23. MKMatt Kaeberlein2:34:49

        Yeah, right

    24. PAPeter Attia2:34:50

        ... a lot of people, Matt, are looking to this study, um, pr- potentially along with the work of Adam Solomon. Maybe we can just touch on that really, really briefly as well as another model.

    25. MKMatt Kaeberlein2:35:00

        Sure. Let me, let me make a comment on that, though, um, which is that I'm not sure that lifespan... So even though we're powered for lifespan, that's our primary endpoint, I'm honestly not sure that's the most important endpoint for evaluating potential efficacy of rapamycin in, in dogs or people, right? I mean, I think we wanna think about this more broadly speaking in the sense that there may be some healthspan metrics that are particularly and potently positively impacted by rapamycin-

    26. PAPeter Attia2:35:29

        I think people just also wanna make sure-

    27. MKMatt Kaeberlein2:35:31

        ... in certain people

    28. PAPeter Attia2:35:31

        ... there's no negative lifespan, though. That's the thing, too, right? It's-

    29. MKMatt Kaeberlein2:35:34

        Oh, absolutely.

    30. PAPeter Attia2:35:34

        Yeah, yeah. So it's like-
21. 2:36:20 – 2:39:31

    Preliminary results of primate studies with rapamycin

    1. PAPeter Attia2:36:20

       that, that would probably be sufficient enough reason.

    2. MKMatt Kaeberlein2:36:22

       Right. Okay. So now Adam's study. Yeah.

    3. PAPeter Attia2:36:25

       Yeah.

    4. MKMatt Kaeberlein2:36:25

       So this is a super interesting study in a non-human primate called a marmoset. Marmosets are, are an interesting non-human primate 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. Uh, 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, uh, in captivity. So they make them-- That makes them an interesting model as a non-human primate, uh, to study aging. And, and so Adam, for several years now, has had an ongoing, uh, marmoset colony, uh, in San Antonio, um, some of whom have been getting rapamycin. And I don't think they've published the data from that, that study, uh, at least not the lifespan data, so I don't think it's, I don't think it's complete. Um, but, uh, but they've already published some preliminary data for, you know, bioavailability, blood levels, some interesting data, suggestive. Um, and Adam has talked in meetings about the survival, 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 for marmosets, that's, that's really important 'cause now it's gotten to the point of a primate, right-

    5. PAPeter Attia2:37:58

       Yeah

    6. MKMatt Kaeberlein2:37:58

       ... 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, you know, there are some questions about rapamycin, particularly, again, we've already talked about, and 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, um, it may be the case that the effects of rapamycin are going to be somewhat different than what we see in the laboratory. So, so it's a huge, 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, a laboratory-based study.

    7. DSDavid Sabatini2:38:44

       To me, one of the big issues with the mouse studies in rapamycin is that these are sedentary mice who are getting-

    8. MKMatt Kaeberlein2:38:50

       Mm-hmm

    9. DSDavid Sabatini2:38:50

       ... fat, who are, who are frankly depressed. And so what, what I always say about your study as the critical aspect is that these are free-living animals who presumably are relatively happy. And so the marmoset study sounds exciting, but it does have that big caveat of sort of-

    10. MKMatt Kaeberlein2:39:03

        Yeah

    11. DSDavid Sabatini2:39:03

        ... potentially more sedentary, sad animals. And occasionally, I actually frankly hadn't even thought about the potential infectious disease implications of it. But, but the, the living in the human environment, to me, is the t- the key aspect that I, that I'm looking forward to your study about.

    12. MKMatt Kaeberlein2:39:22

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

22. 2:39:31 – 2:49:33

    Dosing of rapamycin

    1. PAPeter Attia2:39:31

       What was the dosing in your study, Matt? Is, is it 0.1 milligrams per kilogram weekly?

    2. MKMatt Kaeberlein2:39:37

       No. So we, uh, we've done two. The first one was, was we tested two doses, 0.1 milligrams per kilogram three times a week and 0.05, so half that dose-

    3. PAPeter Attia2:39:47

       Okay

    4. MKMatt Kaeberlein2:39:47

       ... three times a week. And then we went to, uh, 0.15 milligrams once a week, and that's what we're using now. And, you know, we could talk about why we, why we made that decision. This is a 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, and all of that. Um, 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, um, in terms of total dose, so cumulative dose. And we haven't really talked yet about what sort of become popular in the, 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, the, 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, um, but that was... All of those things kinda weighed in to that decision.

Episode duration: 3:01:28