#272 ‒ Rapamycin: potential longevity benefits, surge in popularity, unanswered questions, and more | David Sabatini, M.D., Ph.D. and Matt Kaeberlein, Ph.D.
Episode Stats
Length
2 hours and 50 minutes
Words per Minute
182.21045
Summary
Dr. David Sabatini and Dr. Matt Caberlin are two of the most knowledgeable people on the planet on the topic of rapamycin and MTOR. In this episode, they discuss the discovery of this molecule, the pathways it s believed to drive its impact on lifespan, and why it should be prescribed to patients.
Transcript
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Hey, everyone. Welcome to The Drive Podcast. I'm your host, Peter Atiyah. This podcast,
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my website, and my weekly newsletter all focus on the goal of translating the science of longevity
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into something accessible for everyone. Our goal is to provide the best content in health and
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wellness, and we've established a great team of analysts to make this happen. It is extremely
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and benefits above and beyond what is available for free. If you want to take your knowledge of
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of the subscription. If you want to learn more about the benefits of our premium membership,
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head over to peteratiyahmd.com forward slash subscribe. My guests this week are David Sabatini
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and Matt Caberlin. Matt has been a former guest on three occasions, episode 222 and 175, and all the
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way back to episode 10, while David was on the podcast way back in episode number nine. Not only
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are they two of the original guests on the podcast from our 12-part pilot experiment in the summer of
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2018, they are also two of the most knowledgeable people on this planet on the topic of conversation
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today, rapamycin and mTOR. In my conversation with Matt and David, we cover the discovery of
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rapamycin. We look at how mTOR, which sits at the epicenter of our existence, works and does its job.
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We talk about the pathways of rapamycin that are believed to drive its impact and improvement on
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lifespan. We discuss the initial studies that showed rapamycin may be gyroprotective, and also
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what studies have come out since or are currently in progress, which provide more information and
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clarity around this very important question. Finally, we discuss what is known and unknown about
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the potential frequency and dosing of rapamycin in humans. I'm very excited to release this podcast,
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because I don't think there is a question I get asked more about from my patients than this topic.
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Understandably, because my patients know that I take rapamycin and have been doing so for about five
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years, it's understandable that they want to understand if it's something they should be doing
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too. And I think you'll see from this discussion why I have reservations about just blindly putting
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people on rapamycin. In other words, why is our practice not a rapamycin mill? I hope that this
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podcast is helpful, not just for my patients, but everybody listening, including everyone who is
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interested, of course, in this question. So without further delay, please enjoy my conversation with
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David Sabatini and Matt Cabron. Well, guys, we're going to try something a little different today,
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which is we're going to try to have a three-way discussion, which is something we would easily
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be doing if we were sitting over a meal, but always makes for a slightly more challenging podcast.
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That said, given our familiarity with each other and your familiarity with this topic, I am
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a hundred percent confident this is going to be an amazing episode. It's also an episode that is
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long overdue. So you guys are both in the camp of, I believe, first dozen or so podcasts that were
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released on the drive a little over five years ago. Matt, you and I have spoken a number of times
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since. David, you and I, at least on the podcast, have not, obviously, in person all the time. So with
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all that said, many people are going to be new to this topic. They will have heard a lot about it.
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They may have even read a chapter about it in my book, which you guys were both very gracious to
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help me fact check and edit. But here we are. We're going to pretend that someone coming into
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this discussion doesn't really know anything about rapamycin, doesn't really know what this
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mTOR thing is. I hope that by the end of this discussion, we will have provided people with
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arguably the most comprehensive, quasi-concise explanation of all you need to know about said topics.
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With that said, I would like to ask each of you to do something I don't often ask my guests to do,
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which is toot your own horns a little bit about what it is that allows me to say you are each
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among the two most knowledgeable people on this topic. Let's start with you, David. You've worked on
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this molecule, rapamycin, your entire scientific career going back to your PhD. And here we are
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30 plus years later, you're still the leading authority on it. Can you tell us a little bit
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about that? Sure, Peter. Thank you for having us. Nice to see you, Matt. So no, indeed, I've worked on
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rapamycin my whole life. When I was a student with Saul Snyder or Johns Hopkins, I became fascinated with
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this molecule. And frankly, I needed a research topic. And so I tried to figure out how it works.
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And that led to the purification of protein that we now call mTOR. Michael Hall had identified a
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yeast version of this that Matt was one of the early workers on this called TOR1. And since that time,
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we've done a lot of biochemistry, try to figure out what this protein does. And at the end of the day,
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what we conclude in a kind of big picture point of view is that this is the protein that links
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the availability of nutrients in our environment to whether we're in a catabolic or an anabolic
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state. Anabolism growth, catabolism breakdown of material. And I think that accounts for why mTOR
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has so many different roles. Because if you think about our sort of evolutionary history,
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there's almost nothing in our physiology that shouldn't be controlled by the availability of
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nutrients. It's such a central thing in our lives. We tend to forget that now because of course,
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we're in a overeating stage. And since that time, what we've done is figured out a lot of the pieces
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of this pathway, including what we call two complexes, protein complexes, mTORC1 and mTORC2.
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And really the work that I'm the most satisfied with is how it senses nutrients and the nutrient
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sensors themselves, which are the actual proteins that bind the small molecules that tell mTORC1 in
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particular that it detects nutrients. And so I'm excited to be here and to delve into some of the
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implications of this work. Awesome. Thanks, David. Matt, people who are listeners of this podcast are
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going to be maybe a bit more familiar with you because in addition to that very first podcast
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we did circa 2018, you've been back a number of times and we've talked about mTOR and rapamycin,
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but we've also talked about protein, nutrition, and things like that. But maybe for folks who are
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hearing you for the first time today, can you give a similar bio of what it is that allows me to
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also refer to you as one of the world's absolute leading authorities on this topic?
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Sure. First of all, thank you for having me back. I'm glad you aren't sick of me yet. It's fantastic
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to be here with David. And I wish I could say that I was smart like David was and recognized
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immediately how important mTOR would be and rapamycin. But I actually started working on mTOR
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in yeast kind of by accident. So we were really interested in understanding what are the genetics
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that control longevity. And so we did an unbiased search for new genes that would affect lifespan
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and happened to find mTOR. And when we made that discovery, I immediately went and looked up
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everything I could learn about mTOR and found out there's this drug, rapamycin, that's an inhibitor
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of mTOR. And then we found that we could also increase lifespan with rapamycin. At this point,
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we were working in yeast. But then it became clear to me because of the work of others that this pathway
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and this drug appeared to affect the biological aging process, not only in yeast, but also across
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the animal kingdom. And we now know even in mammals like mice and potentially in larger mammals like dogs
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and people. So I think with that knowledge, I got very interested in trying to understand what the
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mechanisms are for how rapamycin was affecting the biological aging process. We've studied this in
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yeast and worms and fruit flies and mice, a little bit in pet dogs, which we may talk about. And I think
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through all of that, the one thing that has kept me excited about rapamycin as a potential longevity
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therapeutic is that it always works. And I would say without question, it is the most robust and
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reproducible drug, at least from preclinical studies that we know about today for impacting
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not only longevity, but to the extent that we can measure various metrics of health span
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in complex animals, rapamycin also seems to positively impact pretty much every aspect of
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health span that we measure. So I've continued to study it for that reason. And I think probably what
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I'm maybe best known for these days is pushing forward a veterinary clinical trial of rapamycin to really
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start to answer the question of all the things we've learned about rapamycin in the context of
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aging and longevity in the laboratory, how much of that will translate into the real world? And so we are
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actually carrying out a veterinary clinical trial of rapamycin in pet dogs right now. We've got some
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preliminary data, but it's too early to be able to say, you know, with any level of confidence that
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rapamycin is going to positively impact the aging process in dogs. But I think we've already learned a lot
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about safety and maybe some hints about efficacy. So that's pretty exciting. And that's something that I
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quite passionate about continuing to push forward and see where we end up.
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And I guess I should, before I make my next comment, disclose that I and a number of my patients are
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funders of a study that we will undoubtedly talk about. So we should just, I guess, declare that
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as a conflict if people want to consider that a conflict. But regardless, I think what's really
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great about having you guys together, and tell me if you agree with this assessment, is on the
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continuum of understanding rapamycin and mTOR, David, you're closer to what we would call the bench
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side of things. And in many ways, Matt, I consider you kind of closer to the bedside. So people have
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heard this term bench to bedside, i.e. translational research. And obviously the bedside in this case is
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not just the bedside of humans where we aren't quite yet, but really the bedside of more complex mammals.
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So would you guys kind of agree with that assessment that your skill sets and your knowledge
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base and your research are very complementary through that continuum of bed-to-bedside?
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I would. I would add, though, that Matt takes the work in a very serious scientific way, right? And so
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I think in a field where it's very easy to get caught up in boosterism and claims that you see online
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all the time that are extreme, I think Matt has been very careful. And part of what's contributed
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to the interest in rapamycin, frankly, is that Matt has been careful about this. And so he's taken a
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very scientific approach. And as I've told many people who know me well, I pretty much put Matt
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in one of the most respected categories of aging researchers for that reason.
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Peter, let me just give a little bit of a twist on what you said. I mean, I agree with what you said
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conceptually. I would also say, I think even though much of my research on mTOR and rapamycin has been
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what people would typically consider preclinical or basic research, it is different. I agree. I think
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the approach that David has taken throughout his career is quite complementary in many ways to the
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approach that I've taken in that David has really, as you already said, been the pioneer and the leader
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in really understanding detailed mechanistic aspects of the whole mTOR signaling network.
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And I don't know how much we're going to get into that, but I think it's useful for people to
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appreciate that this is an extremely complex network of biological interactions. And there's
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no question that David and his lab and people who have come from his lab have really played the
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leading role at fleshing out from a very detailed biochemical and mechanistic perspective, how that
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network is working. And that has, I think, in many ways laid the foundation for people like me and many
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others who have then taken that knowledge and tried to start to move it into maybe more applied
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That's a beautiful way to describe it. And I also want to echo something that David just said. And again,
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not to embarrass you, Matt, but I do think that the field owes a lot of its credibility to the way you have
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approached it with scientific rigor being the highest priority, as opposed to the commercialization
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not being front and center. And I do think that there are a lot of other molecules that maybe we
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don't have to get into today, where there is some interesting science behind it, but it seems to have
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almost been corrupted by a commercialization route. And the corruption of that has meant, A, we may never
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know if these things work or don't work, but more than anything else, they're very difficult to take
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seriously. And I think everybody should be very grateful for the way the field has gone.
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So before we dive into it, and I know this is a story that's been told before on this podcast,
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and I probably even write about it in the chapter of my book, I do think that the discovery of
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rapamycin is the place to begin this, because there's a very unique phenomenon here, which is
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the drug was discovered before the target, and the target is named after the drug in response to that.
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So David, you and I got to visit this very special place where the bacteria that ultimately
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produced this drug was discovered. We certainly have plans to go back. It's on our list of things
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to do in the next few years, and we shall. Do you want to give folks the story of how this
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Sure, sure. And I hope we'll go back soon, and I hope Matt will come as well.
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For the record, I did try to get this recording done on Rapa Nui, so I just want to put that out there.
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As you know, Peter, there were, in our attempts by pharmaceutical companies to collect soil samples
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and other biological containing samples throughout the world, and their Wyeth Ayers did come into
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possession of a soil sample from Easter Island, otherwise known as Rapa Nui in the South Pacific,
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at one point claimed to be the most remote island of the world. I think it's actually technically not,
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but very far from anything. And in that soil sample, actually in Canada, people eventually
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isolated bacteria from it. Bacteria called Streptomyces hygropagus. And from that bacteria,
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rapamycin, was eventually isolated, and in deference to Rapa Nui was named rapamycin.
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Now, ironically, it turns out when people have looked for rapamycin and other bacteria throughout
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the world, and in fact, even the same bacteria, it actually has been found in many other places,
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but it did come originally from rapamycin. And like was done at the time, these molecules,
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these bacterial products, you really would call an antibiotic, it did come from bacteria,
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were tested, you know, in many different assays. And I actually think, and Matt may correct me,
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I think some of the earlier assays were actually immunological assays, even before some of the
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antifungal assays. And that eventually led, many decades after, to pursuing it as an immunosuppressant,
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but in the meantime, it was also found to have antifungal agent activity. And that's where some
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of the genetics of rapamycin and some of the targets were first identified because of the ease of
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genetics. So this is a story that began, I think the original soil samples may have even been in
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the 60s. Yeah, I think it was 66 or 67 soil samples. And then Seren, I don't think really
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got around to digging into it until 71 or 72. Exactly. And then he championed it. In fact,
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one of my most valued possessions, when I started working on rapamycin, we didn't have much. And Saul
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Snyder, my advisor, wrote Seren and asked for some. He sent us many grams, which I had laid
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or calculated, had a street value of many hundreds of thousands of dollars if one could sell it like
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that. And a really nice note wishing me luck. And the entire bibliography of rapamycin at that time,
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which consisted of his papers and a couple of abstracts, is a little thin book at the time.
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And he is the one who championed it. The clinical path took way too long. And I think that even
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impacted some of its utility because the patents expired, I think, before you could really sort of
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capture some of the value of it. So we're talking about something now that's in the 50-year range
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plus. And I think a question that we could ask ourselves, and I think we will, is rapamycin as
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good as it gets? There obviously are derivatives of rapamycin. But even in this pathway, which as Matt
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says, exceedingly complicated, are there other targets that we should be pursuing that may actually
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have equal or better impacts on the aging process? Can I just add one thought? Something David said
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there we may again also touch on, which is the clinical path not only took too long, but I think
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you can make an argument that the clinical path has actually maybe negatively impacted the development
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of rapamycin and other mTOR inhibitors for other uses. Because it was developed clinically as an organ
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transplant immunosuppressant, and that's how it was first approved, it was used in a dosing protocol
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and a patient context where there are lots of side effects. And I think we are still learning
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what the side effect profile actually looks like for rapamycin at lower doses in patients who are
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not immunocompromised and haven't had an organ transplant. So I do wonder whether the history of
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rapamycin and the rapidity at which it will be eventually tested for other endpoints in clinical trials
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where it may have benefits has been negatively impacted and slowed down because of the reputation
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that the drug got as a dangerous drug based on the way it was developed clinically. So I think that's
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an important piece of the puzzle here to think about. Just to give some numbers to it, the first paper
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that Soren Seagal put out there describing the chemical composition of rapamycin, if I'm not mistaken,
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was about 1971, 1972, the FDA approval for rapamycin in humans was 1999. Just to give you a sense of what
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you're both talking about here in terms of an enormous gap of time between when you sort of make
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a chemical discovery, file an IND and work all your way through. Second point I'd make is as a former
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surgical resident, I was in my surgical residency taking care of transplant patients when rapamycin
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was in full use. Now, again, it's interesting, David, the whole reason you got involved in
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rapamycin was because of FK506, which was a cousin of rapamycin that, if I recall, the whole reason
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your lab was using rapa was as a control that didn't have sort of the calcineric properties of
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FK506. But that's an interesting footnote. But we were giving rapamycin out constantly. And to your
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point, Matt, it was a drug that was typically given two to three milligrams a day every single day
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but with three other drugs. You were also getting prednisone, cell sept, MMF. You were getting
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very, very toxic drugs because you needed to completely shut down the cellular immune system
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of a patient who had just received a foreign organ. And I think that speaks to this point,
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which is for the better part of a decade, 1999 to 2009, the only experience the scientific world has
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with this is in that context. Yeah, you're going to see a lot of side effects, but how do you know
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they're from rapamycin? And how do you know that they would be the same elsewhere? So what happened
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in 2009 that kind of changed this? And David, I'm most interested, I think, in hearing this from you
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because by this point, you've already established your own laboratory. You're working on rapamycin.
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You're working on mTOR probably more so than anything else and trying to understand the nutrient
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sensing pathways around it. But how aware were you of the ITP, the interventions testing program,
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in the buildup to that first study in 2009? I was not very aware of it, I have to say. But I do have
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to say that once we started making that connection of rapamycin to nutrients, which many groups did,
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if you actually look at the history of it, and it was already appreciated for many, many decades before
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that things like caloric restriction had an impact on lifespan. So the idea that rapamycin could have
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an impact on lifespan was one we actually thought of. And we actually, this just tells you how science
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works, we actually tried dosing C. elegans, worms, with rapamycin, naively not realizing that their
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cuticle would not allow rapamycin the way that we were giving it to have an impact. We had found no
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impact. And then there were genetics that came out in worms and Matt's work. And a lot of other people
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really pioneered the aging space, not us at all. But I remember when that paper came out, I think it was
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a nature paper that came out reporting rapamycin as one of the bigger hits in the ITP study. And I think
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what happened there, I think Matt said this before, it connected his work in yeast and work in other
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organisms with a mammal. Now, we just take that for granted, right? Because as Matt said, it does impact
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all those different animals and single cell organisms. But the idea that we had a molecule that spanned from a
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yeast to a mouse was dramatic. That was like a huge, huge impact. Again, we take it for granted now.
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I'd like you to say more on that, both of you. I do think that the evolutionary gap from yeast
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to flies, worms, mammals is a billion years. Are there any other molecules that have done what you
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just said, David? I don't know if there are, but certainly dietary restriction, yes,
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in one form or another. And that did link all those organisms. And as far as I know, it was all done
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before rapamycin, before the discovery of TOR. So there was this universal intervention. I think
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even in bacteria, people have shown impacts on a replicative lifespan. So that, I think, was considered
00:22:07.740
this universal connector. And that's why when the nutrient connection came out, I think we and others
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started thinking along the lines of rapamycin as a mimetic and potentially having this impact.
00:22:17.700
So I don't know, Peter, where there are specific molecules that do that.
00:22:21.700
I'm not aware of any. I mean, yeah, I'm not asking the question rhetorically, but I agree with you that
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outside of caloric restriction, which by the way, doesn't universally extend life. There are models
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and certainly times at which that can be administered when it is not a life extending strategy.
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But yes, I mean, rapamycin in that sense stands alone. Unless, Matt, you can think of a
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counterexample that I'm missing. I'm going to try to respectfully tell you guys that you're wrong.
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So here's what I would say. Rapamycin for a small molecule is probably the only pharmacological
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intervention that has been reproducibly shown to robustly increase lifespan and healthspan across
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that broad evolutionary spectrum. There are other things out there like alpha-ketoglutarate,
00:23:08.900
where there are reports in yeast and worms and flies and mice of lifespan extension. It just
00:23:14.600
hasn't been tested or reproduced as much. But on the genetic side, and this is where I wanted to
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just add a little bit of additional context to what David was saying, which is that with rapamycin,
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it's not only the drug, but we also have genetic inhibition of mTOR in each of those model systems
00:23:32.360
that recapitulates the longevity and healthspan benefits. So it's a rock solid, airtight case for
00:23:39.700
mTOR and longevity. But also on the genetic side, this is a study that we did with Brian Kennedy and
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Daniel Promislow, this was probably 2007, where we asked the question, if we looked at all of the
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genes at that time that were known to affect lifespan in yeast, and all of the genes that were known to
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affect lifespan in worms, and we simply looked at orthologs, meaning the same gene in each organism,
00:24:02.000
how often is genetic control of longevity shared? And it turns out it's pretty often. So there is a
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relatively high degree of evolutionary conservation at the level of genetic control of longevity
00:24:14.020
across a broad evolutionary distance. And that's really been the whole thesis of my
00:24:18.640
career, right, is trying to understand those evolutionarily shared mechanisms of longevity.
00:24:23.160
So I just think it's important for me, I guess, to say that because there's a lot of confusion now in
00:24:28.160
the field. There have been a lot of new people come into the longevity field who, for whatever
00:24:32.240
reason, aren't familiar with a lot of this history. And so they ask questions like, well, how do we know
00:24:38.180
that you can use worms to understand anything about aging in a mammal? And I'm like, because we already
00:24:43.320
know that the genetics of longevity are conserved. Not everything's going to be conserved. But it has
00:24:48.680
been statistically shown that there is a conservation of the biology of aging. And that's kind of
00:24:54.540
fundamentally important to how we think about studying the biology of aging in the laboratory, and then
00:25:01.480
potentially translating those discoveries into the real world. So again, sorry for the tangent, but I do
00:25:06.560
think this is an important, more fundamental biology of aging point that's useful to just reemphasize, because
00:25:14.780
I completely agree, Matt. And when I just saw Peter in Austin, and he took me on a rocking trek in 104 degree heat,
00:25:22.420
we exactly talked about this topic. And my point was that biochemical cell biological processes
00:25:29.900
that are conserved amongst all these organisms are going to be the ones that are going to impact
00:25:35.020
aging. And in fact, I tend to dismiss those processes, which are less conserved as potentially
00:25:41.020
causing impacting the aging process. I 100% agree to you that whatever is the fundamental issue that
00:25:46.800
happens in cells that leads to aging is going to be conserved. And therefore, the regulators of that
00:25:52.180
process, or the impactors of that process will be conserved.
00:25:55.740
I want to come back to this point. If we had been able to record that RUC session,
00:26:00.800
if you could eliminate all the huffing and puffing, it would have been a great podcast in and of itself.
00:26:05.980
And we're going to come back and talk about some of those things. This is actually a great step off
00:26:10.560
to make a point that what we're talking about here is the broad term of Giro protection.
00:26:16.200
And I always kind of differentiate this when I'm talking to my patients.
00:26:18.900
I say there are certain strategies that we take to extend your lifespan and improve your health
00:26:25.500
span that are very disease specific. So for example, the attenuation of apolipoprotein B
00:26:33.160
is undoubtedly going to lengthen your life if implemented for a long enough period of time,
00:26:39.460
and by extension, I would argue, improve the quality of your life. But it's doing so through
00:26:44.680
two disease processes. It's doing it through a reduction of atherosclerotic cardiovascular disease
00:26:51.220
and cerebrovascular disease, but also through all lines of dementia. But it's not attacking a
00:26:57.740
fundamental pillar of aging. It's a very disease specific hack, for lack of a better word. And by
00:27:04.460
the way, it certainly wouldn't be applied to organisms beyond ourselves. Very few organisms
00:27:11.560
have ApoB, i.e. very few organisms succumb to ASCVD. That doesn't mean that we shouldn't look
00:27:18.300
at disease specific tools to modulate lifespan and health span. But what we're talking about here is
00:27:24.900
so much more fundamental. I will not put either of you guys on the spot and ask you if you can recite
00:27:30.340
the nine hallmarks of aging. Matt and I tried that on our last podcast and got... I can get seven,
00:27:35.180
I think. We could do this like name that tune, right? How many hallmarks of aging can you...
00:27:38.940
Right, right. But there are nine of these hallmarks of aging. There's actually 12 now,
00:27:42.860
right, with the new and improved version. Oh, there is. My God, I'm so dated.
00:27:47.640
Okay, so let's now dive into mTOR a little bit. Can we go back to that mouse study?
00:27:54.280
Go back to the ITP. Yeah, sure. Yeah, because there's another part of that study. So just for a little
00:27:59.000
bit of context, I'm not sure this ever got said explicitly, but this was a study from the NIA
00:28:03.460
Interventions Testing Program published in 2009. It was the first study to show that rapamycin treatment
00:28:09.020
in a mouse could extend lifespan. And that was important. But I think the other, maybe more
00:28:14.620
important part of that study that often doesn't get always talked about is that this was the first
00:28:19.640
time that any intervention... You could argue a little bit about caloric restriction. That's kind of
00:28:24.540
a tangent. But I would say it's the first time that any intervention was convincingly shown
00:28:28.840
to extend lifespan when treatment was started in middle age. So about the mouse equivalent of a
00:28:34.200
60, 65-year-old person biologically. And that, again, as David said about rapamycin,
00:28:41.160
we kind of take it for granted today that that's possible. But in 2009, I don't think anybody expected
00:28:47.120
that experiment to work. It was actually an accident that they ended up doing the experiment that way.
00:28:51.820
And it had to do with the fact that they couldn't formulate the rapamycin in the mouse chow in a way
00:28:56.820
that was stable until the mice were already about 12 months old. So treatment started, I think in that
00:29:02.260
case, it was 20 months of age when they finally started the treatment. So it was a happy accident.
00:29:06.980
But in my view, I've said this before, I think this is one of the most important studies in the field
00:29:13.380
in the past 20 years, maybe 50 years, for that reason, that it opened up what we now consider to be
00:29:20.120
routine, which is that you can actually have an impact on longevity and some metrics of health
00:29:26.560
span when you start treatment in middle age. And as we have started as a field to think about
00:29:31.180
translational application, that becomes hugely important because suddenly we're talking about
00:29:35.480
treating middle-aged dogs or middle-aged people as opposed to trying to treat puppies and teenagers.
00:29:40.800
And that's just much more pragmatic and practical from the perspective of actually being able to
00:29:45.140
implement. Especially when you consider what David said at the outset, which is mTOR is the
00:29:50.320
master regulator of how nutrients trickle into the system. Are you going to be in an anabolic state or
00:29:57.080
are you going to be in a catabolic state? Well, Matt, you'll be pleased to know we just got a puppy
00:30:01.100
recently. So we've got this adorable little three-month-old puppy. And I don't think it would make sense to
00:30:08.640
necessarily inhibit mTOR in an animal that is purely about anabolism right now. It's trying to grow.
00:30:16.020
And it would be suboptimal if we had a therapy that we believed could only work if administered
00:30:21.940
early in life. And yes, your telling of that story is remarkable. And I think also speaks to the
00:30:27.840
serendipity that often lies in scientific discovery. It's often an accident or something going wrong that
00:30:35.220
leads to that. And I've talked about this, I think, with Rich Miller. They were contemplating
00:30:39.560
sacking the whole study because they couldn't get the rapamycin formulated.
00:30:44.680
It is a fascinating question, though, why the starting point of delivery of rapamycin does have
00:30:50.840
an impact on the life extension and health span extension. And with the biological basis of that
00:30:55.740
is something that at least I don't have a great conceptualization of that. And I'm sure Matt has
00:31:01.560
thought about this much more. But it's interesting to think about how one designs experiments to try
00:31:06.420
to ask that question. Is it safe to say we don't know when the ideal time to implement would be?
00:31:13.480
I think it depends a little bit on what you mean by ideal, right? So this now gets into
00:31:17.540
risk-reward and side effects versus benefits. In mice, we absolutely don't know in terms of lifespan.
00:31:23.660
If we take that as the primary metric that we're interested in, we don't know when is optimal to
00:31:29.600
initiate treatment or what dosing protocol is optimal. So there still has not been a full,
00:31:35.460
what I would call even dose response profile of rapamycin across a single intervention time
00:31:42.440
point, initiating time point. The answer is no. And honestly, I think we probably never will simply
00:31:47.720
because the cost of doing those experiments and all the permutations that you could come up with for
00:31:53.380
time that you initiate and different doses to test, I just don't think anybody would ever
00:31:58.740
fund that study. We're getting off on a tangent, but it's probably worth just mentioning that going
00:32:04.640
back to the starting in middle age, this is where I actually have some real concerns with the way we
00:32:09.520
fund biomedical research in general. If somebody went to the NIH before this study had been completed
00:32:15.600
and said, we want to start an experiment with rapamycin starting at 20 months of age in mice,
00:32:20.700
that grant never would have gotten funded because people would say that'll never work. And so this is
00:32:25.640
where I think, again, it was very fortunate in this case that it happened the way that it did,
00:32:30.700
but I would argue as a research enterprise should develop an appetite for higher risk, higher reward
00:32:38.020
projects. And I don't think anybody's going to disagree with that, but I think this is a nice
00:32:42.020
case in point of an important discovery that changed a field that would not have been made
00:32:47.300
if not for just the fortuitous circumstances that happened.
00:32:52.360
Yeah, I think that's completely fair. I would challenge you on one thing though, Matt. I can't
00:32:58.080
think of a better type of research to fund for relatively low dollars than the types of questions
00:33:05.720
that you're asking. In other words, I agree that there are a lot of permutations, and I agree that
00:33:10.740
we're talking about tens of millions of dollars. But when you consider what's at stake, i.e. what we
00:33:16.780
could learn, I guess for the listener, it's worth explaining something. We're going to come back and
00:33:20.900
talk about this, and we're going to talk about intermittent dosing. But these ITP studies are
00:33:25.440
dosing rapamycin every day. It's mixed into the chow. So the mice are constantly nibbling on a low dose
00:33:33.340
of rapamycin. And what we're going to go on to talk about as we start to extrapolate into, for example,
00:33:39.540
companion dogs and ultimately humans, is a dosing regimen that looks completely different. Well,
00:33:45.220
for starters, I sure as heck would like to see what that looks like in the mice of the ITP. I'd also
00:33:51.100
like to see some of these different permutations around the different, not just doses, but starting
00:33:57.280
points. And again, if it costs 10 million to do that study, I got to tell you, I think we could raise
00:34:02.560
that money. It didn't take too long to raise half that money to do the dog aging project. So I think
00:34:08.600
there would be a real appetite to do that kind of work because the implications are enormous.
00:34:13.800
The NIH might not fund it, which is probably what you meant.
00:34:16.480
That's right. I think also, and we may get into this as well, there are a bunch of those kinds
00:34:20.420
of fundamental questions that I would argue are relatively low-hanging fruit. And then we would
00:34:25.000
have to think about prioritizing. I think we're going to talk a little bit about rapalogs or other
00:34:30.160
classes of mTOR inhibitors. The other classes of mTOR inhibitors, there was just recently the first
00:34:35.400
study that I know of that tested an ATP-competitive mTOR inhibitor in mice. It's intriguing, I would
00:34:42.340
say, early data. But we really have no clue, as far as I can tell, how other classes of mTOR inhibitors
00:34:49.980
would perform relative to rapamycin. That's another super important question that, again,
00:34:54.440
for frustrating reasons has been very hard to get those kinds of studies funded. And I can just tell
00:34:59.400
you from my own experience, I have put in grants to study dose-responsive rapamycin, different
00:35:05.380
intervals of rapamycin testing, and other classes of mTOR inhibitors. And they have been uniformly
00:35:11.920
rejected because, by and large, NIH study sections just aren't interested in funding those kinds of
00:35:18.500
studies. They're not considered, at least in my view, they're not considered mechanistic enough.
00:35:22.980
So I agree completely with you, but I think those kinds of studies will not be funded in the current
00:35:28.420
structure for research funding, even though they're super important.
00:35:32.680
Yeah. So David, let's now start to talk about the how. I think it's worth doing this in a little bit
00:35:39.640
of detail. And I know that the next few minutes might be among the most technically, perhaps challenging
00:35:45.340
for a layperson to understand. But I think it is important to have some understanding of the
00:35:51.420
biochemistry of what this molecule does and what this protein complex looks like and what the cascade
00:36:00.160
of events are that move on. And I think it's also important to understand how nutrients work. So we're
00:36:06.280
going to talk a lot about amino acids and probably in particular leucine. So in any order that you feel
00:36:12.360
it makes sense to walk us through that, David, explain how this molecule, mTOR, which sits at the epicenter of
00:36:23.200
our existence as living entities on this planet, how does it do its job?
00:36:29.620
One thing for the listeners to understand is that rapamycin is quite unique in another aspect that we haven't
00:36:36.220
talked about, but also was very exciting at the time, right? Rapamycin, unlike most drugs, most drugs go
00:36:42.560
and find their protein target and do something, usually inhibit that target. Rapamycin gets in the cell,
00:36:48.660
binds to a little protein, FKBP. What it does to FKBP, frankly, doesn't seem to matter at all, but instead
00:36:56.620
hijacks that protein and now takes it and makes it bind to mTOR. It basically uses it as this thing that it
00:37:05.420
draws next to mTOR and that moving of FKBP to mTOR is actually critical for how rapamycin acts.
00:37:12.560
As people like Stuart Shriver have pioneered, it's really a molecular glue that connects mTOR and FKBP
00:37:18.340
and that interaction is absolutely critical. So how does mTOR work? When we first and others found mTOR,
00:37:24.920
it was this big protein. It looked like a kinase, that is, it's a protein that puts phosphates onto other
00:37:31.480
proteins, but yet what it did, what its targets were, were completely unclear. And I think as we
00:37:37.780
were talking in the pre-session, Matt pointed out, it's incredibly complicated. It probably acts on
00:37:42.280
hundreds of other proteins. In general, what are those other proteins? They're either proteins
00:37:48.420
that make the cell build things, this anabolism side, or break it down. And on the breaking down
00:37:54.840
side, as you and I, Peter, have discussed, I'm sure Matt agrees, autophagy, right? The self-eating
00:37:59.600
and destruction of parts of the cell, sometimes aged parts of the cell, sometimes parts that are
00:38:05.340
damaged for other reasons. That seems to be absolutely critical on the catabolic side.
00:38:11.200
And the way mTOR works, for a long time we had mTOR. We couldn't really get it to phosphorylate
00:38:16.680
anything in a test tube. It just didn't work. It seemed like a terrible kinase. That is,
00:38:21.420
its enzymatic activity was so puny. We even thought maybe it's not really a kinase. It really was like
00:38:26.560
a moribund protein. And the critical breakthrough was the idea at some point that mTOR must work
00:38:35.040
by being bound to other proteins. Now, again, this seems obvious. Everyone talks about the
00:38:40.060
TOR complex, but at the time it wasn't. And the reason was that, of course, we and others had looked.
00:38:44.460
We'd said, okay, isolate mTOR. Does it have friends? And the answer was no. It has no friends.
00:38:50.060
What we came to realize, and this goes back to serendipity, it turns out the detergents,
00:38:56.140
when you have a mammalian cell that's surrounded by a lipid fatty membrane, you have to break that
00:39:02.400
to do biochemistry. It turns out the detergent we were using, which was the most commonly used
00:39:07.560
detergent to break cells, for simply bad luck reasons, broke apart the mTOR complexes. You can
00:39:14.040
never predict this. And why does it? We don't know. And when we moved to other detergents,
00:39:18.860
used things to stabilize it, we then found these TOR complexes. And the first breakthrough for us
00:39:24.560
was the discovery of a protein that got this name, Raptor. At the time, people didn't like this name,
00:39:30.020
but now it's a well-studied protein. And as Matt alluded, there's actually genetics on Raptor that
00:39:34.720
connect it to lifespan and the aging process. And so that defined what we now call TORC1 or mTORC1.
00:39:42.340
Another protein that we named Richter defines what we call mTORC2. I'm sure we'll talk about mTORC2.
00:39:48.480
at the time. And so we started building out that complex. And now when you had that thing
00:39:53.760
in a test tube, it did stuff. It could show serious activity that you could measure. It could do serious
00:39:59.420
phosphorylation. The known substrates, like S6 kinase, that before we couldn't phosphorylate S6
00:40:05.760
kinase to save our life inside a test tube. Now, all of a sudden, you really could. So it really opened up
00:40:11.580
the door. And then that connected mTORC1 to all the other things that in sort of a biological
00:40:17.840
lingo we call upstream. All the proteins that communicate to mTOR, bring signals to it, are
00:40:23.560
upstream of it. The things mTOR acts on are downstream of it. And we've actually done very
00:40:28.820
little downstream, I would say. We really focused on the upstream. I would say the next big conceptual
00:40:35.340
breakthrough for us came when we looked inside of cells and saw that mTOR was in a particular place.
00:40:42.920
And this is an organelle called the lysosome. The lysosome is sort of the recycling center.
00:40:48.240
This is where a cell takes things and breaks them down and releases nutrients. And so
00:40:52.380
it turned out that mTOR lived at this very interesting interface where the cell produces its own nutrients
00:40:59.480
by breaking down things, and also where the nutrients are coming in from the outside at
00:41:04.260
that intersection. And we went on then to find lots of the pieces that allow that nutrient sensing.
00:41:08.840
And I'm sure we'll get into amino acids and other nutrients afterwards.
00:41:12.680
David, if I can interrupt for a sec, approximately how many mTOR complexes exist in a typical cell? And
00:41:21.900
let's talk about maybe what the typical cells are. What's the distribution of mTOR concentration
00:41:27.400
across different cells in the body? Things like that.
00:41:30.680
In terms of numbers, we're talking certainly thousands of complexes in existence. It's not
00:41:35.160
an amazingly rare protein. It's not incredibly abundant at all. You know, it's probably in the
00:41:41.680
hundred to a thousand fold less than some of the most abundant proteins in the cell. The proteins are
00:41:48.020
much, much less abundant than that. And it's distributed actually quite evenly between mTOR 1 and 2,
00:41:53.880
at least in the cells that we have looked in culture. When you look across tissues in a mouse
00:42:00.080
or a rat, it's actually pretty even across tissues as well. And so, to some extent, that puts it in the
00:42:06.180
what sometimes pejoratively is called a housekeeping protein.
00:42:11.480
Exactly. Some of the most important proteins in the cell. What we have found now, and I think
00:42:16.000
others would agree, is that regulation of mTOR levels itself doesn't happen that much. It does,
00:42:22.260
but it's not the critical regulatory input. It's all the upstream stuff. And the regulation of that,
00:42:27.840
that really is where the pathway gets fine-tuned in different cells to different inputs. And where I
00:42:33.660
think we have to start thinking about also for new modalities to target mTOR.
00:42:38.240
We'll park this idea of tissue specificity down the line, but if I'm hearing you correctly,
00:42:43.900
even though I don't know that people have sampled the CNS of humans, based on what we know
00:42:49.300
from mice and rodents of rats and things like that, we have reason to believe that you would have
00:42:55.740
comparable mTOR concentrations within CNS tissue, peripheral tissue, probably everything. I'm guessing
00:43:03.400
virtually everything except a red blood cell or maybe even a red blood cell. Do we know if it's
00:43:07.440
in the RBC as well? There actually is some in RBCs, which has been very confounding to us because
00:43:12.840
RBCs don't have things like lysosomes in them. There's even some in platelets.
00:43:17.980
I've actually always wanted to go and look in RBCs for this reason. As far as we can tell,
00:43:21.800
every cell has some mTOR and mTORc1 in it. And I would argue, and I'm not sure if I'm 100%
00:43:28.280
correct in this, I would argue that in almost every cell, mTORc1 is a very critical protein for the
00:43:33.200
health of that cell. And Matt alluded to a study, I guess, where people have used now catalytic
00:43:38.560
inhibitors. And we need to distinguish that what rapamycin does, people call it an allosteric
00:43:43.780
inhibitor. It binds to mTOR, but it doesn't bind in the heart of mTOR. If the heart is where it does
00:43:51.800
its phosphorylation reaction, that's sort of like the central node of it. It doesn't bind there. It
00:43:57.780
actually binds close. And what it does, it prevents certain substrates from getting to that kinase
00:44:03.840
domain. It kind of hysterically blocks them from getting there. So it doesn't fully inhibit all
00:44:08.820
the activities of even mTORc1. So let's give people an analogy, David. So for example, in this case,
00:44:14.500
if the amino acid is like a baseball that's supposed to bind inside the glove, rapamycin by blocking that
00:44:21.700
doesn't sit itself right in the heart of the glove, it maybe binds outside the glove and closes the
00:44:28.060
glove. It changes the shape of the glove so that the intended target doesn't. Is that a good analogy?
00:44:33.480
It is. Now the thing that binds in the glove here is ATP, which is the phosphate donor, and then the
00:44:38.100
substrate, let's say S6 kinase. You're exactly right. ATP can get in there no problem. It's small. It can
00:44:44.380
easily get there. But what happens is basically, it's almost like the entrance to a cave. And now you've put a
00:44:50.040
boulder in the entrance of that cave, but you haven't fully blocked that entrance. So simplistically
00:44:55.980
speaking, some small things get in there. Some smaller substrates can get in there, but some
00:45:00.460
bigger ones can't. And there's also, of course, as you alluded to, shape changes and stuff. But the
00:45:05.140
simplest way to think about it, it's a steric block of some things, but not others.
00:45:09.980
Perhaps also worth just re-mentioning that this is the mTORc1 cave, which is again different from the
00:45:15.720
other classes of inhibitors, which are going to affect mTOR in both mTORc1 and mTORc2.
00:45:22.660
And Matt, you said that there's been a study now on lifespan, or at least aging writ large,
00:45:27.240
with catalytic inhibitors. This is actually something I've always wanted to do because
00:45:30.840
they're extraordinarily toxic molecules when dosed at higher levels. So I'll be curious.
00:45:35.920
I've not seen this, but you're right. The catalytic inhibitors basically annihilate the activity
00:45:41.580
of mTORc1 and mTORc2 if used at the right dose. Rapamycin partially inhibits mTORc1 and over time
00:45:48.500
can also partially inhibit mTORc2. So they're very dramatically different.
00:45:53.120
Can you say a bit more about that latter point? It's a very subtle point, but it's going to come
00:45:57.900
up again when we talk about the difference between continual dosing and intermittent dosing.
00:46:03.540
What is it about the kinetics of rapamycin's inhibition of mTORc1 that will eventually but
00:46:11.840
not immediately lead to the inhibition of mTORc2? Before I'll say that mTORc1, its canonical substrate
00:46:18.520
is S6 kinase. So every biologist looks at S6 kinase phosphorylation as an indicator of mTORc1 activity.
00:46:26.260
The canonical substrate for mTORc2 is a protein called AKT. Everyone looks at AKT phosphorylation
00:46:33.760
as a canonical output. And so I had this postdoc, one of the more colorful people I had, a guy from
00:46:39.580
Kazakhstan actually, Doss Sarbosov, who had discovered Richter and the AKT phosphorylation. One day he comes
00:46:45.820
to my office, he's like, David, rapamycin inhibits mTORc2. And I was like, Doss, that is impossible
00:46:51.620
because we had tried to show that this FKVP rapamycin would bind
00:46:56.260
to mTORc2. And it wouldn't bind. It would bind fine to mTORc1, but it wouldn't bind to mTORc2.
00:47:01.840
He comes, he shows me data. He's like, look, if I use rapamycin for a long period of time,
00:47:07.060
I inhibit AKT and I also break apart mTORc2. And I didn't believe him, frankly, at all.
00:47:15.680
This would have been early 2000s and somewhere in that range. I'd have to look back maybe 2003,
00:47:23.160
2004, in that range. I think we published the paper maybe in 2005. But this is one of those
00:47:28.640
cases, which I'm sure Matt has experienced many times too, where the trainee really is driving
00:47:33.600
the story and convinces you of what turns out to be a pretty important discovery, but I didn't believe.
00:47:39.820
And so the reason was, why did this happen, right? Because you could take mTORc2, you could put
00:47:44.820
FKBP rapamycin on it, and mTORc2 would phosphorylate AKT, no problem. It didn't care. Totally fine.
00:47:50.820
You do the same experiment with mTORc1 and S6 kinase, and now you could really inhibit S6 kinase
00:47:57.260
phosphorylation. So what we came to realize though, and to some extent it's obvious, is that of course
00:48:03.400
mTORc2 is not born as mTORc2. It's born as mTOR and Richter, and they have to find each other.
00:48:11.820
But what happens is that FKBP rapamycin can bind to mTOR, what we call naked mTORc. It can bind to it.
00:48:20.920
And when it's bound, it turns out the Richter can't bind. So you can't make mTORc2. And so what
00:48:27.280
happens then, Peter, is that when you incubate a cell and a mouse over prolonged periods of time of
00:48:33.620
rapamycin, all your mTORc2 acquire an FKBP rapamycin, and therefore you can't form a Richter
00:48:41.640
complex. And so the way that we're preventing mTORc2 formation and therefore mTORc2 inhibition is
00:48:49.000
completely different than how it impacts mTORc1. It's basically preventing the biogenesis, the
00:48:55.020
formation of mTORc2. So you need these two proteins, mTOR and Richter, to come together.
00:49:00.540
Basically, FKB rapamycin is preventing that interaction. And the way people are getting around
00:49:05.980
this, which I think we're going to discuss, is by understanding that at a better biophysical level,
00:49:13.920
So Matt, given what David just said, does it surprise you that the ITP study and many of the
00:49:21.880
studies that have looked at constitutive dosing of rapamycin have still managed to find a longevity
00:49:30.300
benefit? No, it doesn't surprise me. But I think the reason it doesn't surprise me is in part, I think
00:49:36.740
we need to, again, recognize that this network is extremely complicated. So the model that David laid
00:49:43.240
out, I think, is kind of our best guess for how this is working. And I agree, everything he said is
00:49:49.160
correct from a biochemical perspective. What the impact is on the overall network of transient
00:49:56.360
rapamycin treatment at a given dose versus chronic rapamycin treatment at the same dose or a different
00:50:01.240
dose is much harder to really understand in a detailed way. So part of the reason why I'm not
00:50:06.500
surprised is because we kind of already knew all the longevity outcomes before we understood this
00:50:12.560
biochemical mechanism. And so now we're trying to work backwards and say, how do we explain the fact
00:50:18.900
that rapamycin can increase lifespan and a bunch of healthspan metrics, given that the way it was dosed in
00:50:25.740
the mice, should have also impaired mTOR complex too. And built into that is the assumption that
00:50:31.240
the reason rapamycin is extending lifespan and affecting healthspan metrics is purely because of
00:50:36.700
the mTORC1 inhibition. And I would say that piece we don't completely know. The best evidence for the
00:50:42.560
idea that the benefits of rapamycin come from mTORC1 inhibition is the genetic data, which we've sort of
00:50:49.480
alluded to in yeast and worms and flies and mice, where you can mutate proteins or genes that code
00:50:56.520
for proteins in mTORC1 and see lifespan and healthspan benefits. But that's incomplete. So I guess it's all
00:51:03.040
to say that I think, and this is dissatisfying to me and probably everybody else out there, but I think
00:51:08.960
it's true that we still don't fully understand the mechanisms by which mTOR inhibition and rapamycin
00:51:15.880
can impact the biology of aging. And therefore we're working with incomplete models. And I'm not
00:51:22.560
convinced at this point that the idea that all of the benefits are due to mTORC1 inhibition and all of
00:51:29.320
the side effects are due to mTORC2 inhibition, I'm not sure how accurate that model is. It's a model that
00:51:34.440
still needs to be studied. So I completely agree with Matt. I think that last statement is 100% true.
00:51:40.540
I think we almost have no evidence to make that decision one way or another. But I think the
00:51:45.400
reason, if mTORC2, its inhibition is toxic, which we have published papers arguing it is,
00:51:51.760
the reason that I think it's actually quite tolerated is because, you know, in general,
00:51:56.300
the amounts of rapamycin used in the longevity studies are relatively modest.
00:52:01.260
They probably still are somewhat intermittent, even though a mouse is eating them, right? Because of
00:52:06.220
course, it doesn't eat all the time. Unlike what we were doing experimentally, where we were dosing
00:52:10.860
rapamycin very high, keeping it above a certain level. And certainly in tissue culture, it's 24-7.
00:52:16.540
And you can imagine that once an mTORC finds a Richter, it's immune to rapamycin now. So as soon as
00:52:23.940
one of those guys interact, you're going to have an mTORC2. And you need very little mTORC2 to keep
00:52:29.320
AKT happy. We found that early on. You only need probably 10 to 15%, at least in cells and culture,
00:52:34.840
to keep AKT happy. So there's going to be escapers. As soon as rapamycin goes below a certain amount,
00:52:40.540
there'll be escapers and you'll make an mTORC2. I do think we have to ask how relevant that activity
00:52:46.820
is to the potentially beneficial effects of rapamycin. And a lot of the drive to find
00:52:53.080
rapamycins that don't do that comes from my work, right? And so to some extent, I'm saying,
00:52:58.480
hey, look, is that oversold? I think that is a potential argument to make.
00:53:03.980
And Matt alluded to it. I would almost argue there's no perfect experiment to answer this
00:53:07.960
question because mTORC is shared. You almost can't answer this. Almost a philosophical issue.
00:53:15.260
One thing I want to add, David kind of said this, but I think it's really important for people to
00:53:18.600
appreciate. Because sometimes we get into the routine of talking about mTORC and mTORC1 and mTORC2
00:53:24.640
as if they were on-off switches, but they're not. They're kind of like, you can think of them as
00:53:28.060
knobs. And so what David said about you don't need a lot of mTORC2 activity to survive. And the same
00:53:35.280
thing is probably true for mTORC1, but rapamycin is turning down mTORC1 immediately a lot. And that's
00:53:41.660
going to depend on the dose of rapamycin that we give. And then over time, turning down the mTORC2
00:53:46.700
knob, but it's not going to zero. And so again, it's important that people appreciate that it's not
00:53:51.720
been on-off. And that's part of what makes it really hard to do the definitive experiment
00:53:56.180
that David was saying we can't really do given the tools we have because it's so complicated.
00:54:01.500
And the tools we've got are not clean in that context, even though they're very biochemically
00:54:06.200
clean. And there's tremendous feedbacks, Matt, that fight all of that. The system always is trying
00:54:11.500
to get to homeostasis. So David, talk a little bit about discoveries that were made in your lab
00:54:17.260
about what the amino acids were doing to mTORC2. Because those actually are things that were
00:54:23.200
learned much later than the initial discoveries you made around the interaction between rapamycin
00:54:28.960
and mTORC2. So what do we know in particular about branched-chain amino acids or leucine in particular?
00:54:34.080
So this also has a little bit interesting backstory. So when I first identified mTORC2 in Saul Snyder's
00:54:38.700
lab, I talked to my dad, who is a cell biologist. And he said, David, you have to localize mTORC2 within
00:54:45.600
the cell. To be honest with you, I kind of dismissed that in maybe a little bit an arrogant
00:54:49.960
way because I was like, look, I'm a molecular biologist, biochemist, cell biology is kind of an old
00:54:54.240
thing. But we did actually make an antibody to mTORC2. And, you know, at the time we used to make them
00:54:58.920
ourselves in rabbits. We had some, we purified, and I added it to cells. And it gave this very
00:55:04.620
interesting punctate pattern inside the cell. And I remember walking around the cell biology department
00:55:09.860
at Johns Hopkins Medical School, asking people, what is this? And I didn't get any definitive
00:55:15.780
answer. Then the rabbit died, the antibody was lost. And literally for about, that would have been
00:55:22.180
in 93 or something, literally until probably 10, 15 years later, we did not revisit this question.
00:55:29.600
It was Tim Peterson in my lab who did. And when he did do this, and he did it in a definitive way,
00:55:34.600
the answer was lysosomes, as I mentioned. Sorry, just to make sure we understand, David, you're saying
00:55:40.140
when you did the original experiments with the antibodies and they lit up and you're walking around
00:55:44.300
showing them to people saying, what would light up in this pattern? It wasn't clear what the answer
00:55:49.680
was. In other words, it wasn't clear where mTOR was. They saw dots inside the cell. Now it was clear
00:55:55.800
that there was little vesicles. And I think probably, you know, if I had sat down, this was like literally
00:55:59.940
walking around the hallway. So maybe if I had sat down with more experts and really showed them
00:56:04.400
more experiments, we would have gotten a more definitive answer. But that didn't work. And then,
00:56:08.000
you know, you go on. And again, literally the rabbit died, the antibody disappeared.
00:56:12.100
I would say no good antibody to do this experiment for the next 15 years.
00:56:16.840
And this guy, Tim, got one. And he showed, again, we saw the same punctate pattern that I had seen as
00:56:23.740
a student 15 years earlier. But he then went on to figure out what it was. And there were these
00:56:29.020
things called lysosomes. Again, these sort of recycling centers. These are compartments in the cell that
00:56:33.900
have a membrane. Things get in them. And there's about 60 enzymes in that compartment that can
00:56:39.040
basically annihilate anything. Break it down into single components. Like, for example, proteins,
00:56:45.220
amino acids come out. Polymers of sugars, individual sugars come out. And that was fine.
00:56:51.160
But the critical experiment and the one that really changed everything for us is then Tim did a simple
00:56:56.140
experiment. He said, well, let me remove amino acids and look where mTOR is. And it turned out it
00:57:01.040
wasn't on lysosomes anymore. It went off the lysosome. Then he added amino acids. And he had
00:57:06.060
even little movies. Within minutes, it went back to the lysosomes. And so what that told us is that
00:57:12.420
nutrients communicated to mTOR. And one of the things they did was move mTOR to the surface of
00:57:19.240
the lysosome. And then we went on and we found the docking station. So it turns out, you can think
00:57:24.440
of mTOR as like this big ship. And there's this docking, like a pier. And when it gets there, it
00:57:30.340
sits on top of these proteins that hold it there. And it turns out that those proteins are the ones
00:57:37.640
that nutrients talk to. And there's an entire set of proteins about, I think I counted, there's about
00:57:42.700
20 proteins involved in making that communication to drive mTOR to the surface of the lysosome.
00:57:50.200
And we could go into the details of this, but it's probably a little bit
00:57:53.580
too much. But there's multiple large protein complexes that do that communication. And what
00:58:00.420
I think that indicates, and I've said this in talks, could have been simple, right? There could
00:58:04.740
have been one protein, binds an amino acid, talks to mTOR, but it's not. There's a lot of protein real
00:58:10.960
estate used to do this, which tells you the cell cares about this. So the question becomes which
00:58:16.200
amino acids. And I have to say that really, that was broken open, not by us, but by Joe
00:58:22.180
Averick. He had a paper in JBC where he looked at amino acid regulation of mTOR. This was before
00:58:28.900
the lysosomes. He was looking at the activity of using S6 kinase. And he basically found a couple
00:58:34.940
amino acids that matter. He found leucine, a very common essential branch chain amino acid,
00:58:40.240
an important component of whey protein, for example, that people take. Arginine, a very basic
00:58:47.320
amino acid, technically not essential, lots of nitrogen in that amino acid. And those were the
00:58:52.800
two big ones that he found. Now, since then, we have found others. And to us, the holy grail was,
00:58:59.620
how is leucine detected? That was the thing we wanted to know literally for decades. And the reason was,
00:59:07.160
is that there's a lot of literature in mice, in humans, in big animals used in farms, that leucine
00:59:14.360
does cool stuff, like boost satiety, feeling of having fed, boost muscle mass. And eventually,
00:59:21.360
we found it. We found the receptor for leucine. It's a protein called cestrin. And for me, you know,
00:59:26.540
you have in your scientific career, I think you only have a couple of moments where you're kind of moved
00:59:31.580
because you see something and you've been hunting it for a long time and you see it. And for us,
00:59:36.700
was the crystal structure of leucine bound in cestrin, where you're like, okay, this is how
00:59:42.500
nature does it. So from eating a steak to now detecting the leucine in that steak, there it is,
00:59:49.520
it's nestled in there. And then you could sort of imagine how it goes on to talk to emtor.
00:59:57.120
So Rachel Wolfson and Lynn Chantranopong, they had discovered cestrin as the sensor for leucine,
01:00:03.400
and they could show that genetically, biochemically. And then Bobby Saxton,
01:00:07.580
working with us and Thomas Schwartz, he then did the crystal structure of leucine bound. And what was
01:00:13.540
beautiful about that structure was it immediately said, it's got to be leucine, which we and others
01:00:18.560
had shown already, right? You could try isoleucine, but it didn't work. And so you could see it nestled
01:00:23.800
in there and you could see all the parts of cestrin that said, it's got to be leucine.
01:00:27.720
The sobering part was, it's a small little pocket. Leucine is a small molecule, very small
01:00:33.940
molecule. And so it's not clear how you can mimic. The immediate idea was, hey, can we mimic the
01:00:39.740
anabolic effects of leucine without taking leucine? Can we make something better than leucine? And
01:00:44.480
we've managed to make things slightly better, but nothing dramatically better. And the structure
01:00:49.820
tells you why, because it basically is made to fit leucine and nothing else.
01:00:56.440
We don't know, Peter, but it's an interesting question because the pocket,
01:01:00.500
there's a little pocket and leucine binds, and then there's a lid that falls on top.
01:01:05.020
So it literally closes it. The evidence suggests that getting leucine in is easy. Getting leucine
01:01:11.460
out is not easy. And that there actually may be an active way of getting leucine out. That lid has
01:01:18.240
some very interesting sequences in it that suggest that it might be phosphorylated to sort of pop it open.
01:01:24.100
So we don't have an answer to that question. But I think you hit upon something that suggests that
01:01:29.280
it's not the leucine is popping in and out. It pops in, but probably getting out requires an active step.
01:01:37.680
So Matt, how do we reconcile two things that seem a little bit at odds here? On the one hand,
01:01:46.140
we've just established that mTOR is the most important sensor we have, not just for nutrients,
01:01:55.320
but perhaps more importantly, the most critical nutrients of them all, which are amino acids.
01:01:59.980
We also understand that sarcopenia is an enormous risk to both lifespan and healthspan. Sarcopenia
01:02:11.160
meaning low muscle mass. So we understand the relationship between amino acids and muscle mass.
01:02:17.540
We understand anabolic resistance in an aging population. So all of these things say amino acids
01:02:23.600
are good. MTOR activation, i.e. anabolic activation is good. And yet we've just made a very compelling case
01:02:34.340
for why blocking that extends lifespan. How would you start to reconcile what seems conflicting?
01:02:44.160
Obviously, it's going to be extremely complicated. I think I'd start by going back to a point that I made
01:02:49.140
five minutes ago or so, which is that these are not on-off switches. So you really need to think
01:02:54.440
about this in the context of what is the optimal level of mTOR complex one activity for whatever it
01:03:04.100
is that the cell, the tissue, the organ, the organism needs to do to function or stay alive. So certainly
01:03:11.280
we know that you need mTOR activation to build new muscle. And so the idea was that rapamycin treatment
01:03:20.660
inhibiting mTOR, turning down mTOR should lead to faster muscle loss. That was the prediction that
01:03:25.940
was made so that rapamycin should induce sarcopenia if you were to treat animals with rapamycin as they
01:03:32.580
were getting older. That was the prediction that was made. The reality turns out to be the opposite,
01:03:36.680
that it seems to be the case, certainly in rats, probably in mice. We don't have data yet in people,
01:03:42.620
frustratingly, but certainly in rodents that you can treat them with rapamycin throughout adulthood
01:03:48.020
and actually preserve muscle mass into old age. So the explanation for that, I would say, is still
01:03:54.020
a little bit unclear. Almost certainly it's going to be at least partly dose. If you were to give them
01:03:59.160
too much rapamycin, you probably would, in fact, accelerate sarcopenia. But at the doses that have been
01:04:04.880
used to increase lifespan, it seems like you can actually preserve muscle mass during aging. That's a
01:04:11.040
different question, though, than I think, which is one that a lot of people ask, which is, if you were to
01:04:14.980
take rapamycin, would it prevent your ability to build new muscle mass? And it might if you're a bodybuilder.
01:04:22.160
I don't think we have any data in humans on people who are just doing resistance training in the context of
01:04:27.840
just wanting to maintain muscle mass or build a little bit of muscle mass as they're getting older. I just don't think
01:04:33.140
we have that data. And I don't think we have the data in rodents to really answer that question
01:04:37.280
either. In the context of the doses that extend lifespan, would that impair the ability of those
01:04:44.440
animals to build muscle mass if they were put on some sort of a resistance training regimen? I don't
01:04:49.200
know that anybody has done that experiment yet. Which is a shame because it's been done with
01:04:53.600
metformin. There's no reason we shouldn't know that question, right? Sort of. So metformin,
01:04:59.040
you're talking about in human studies? Yeah, in the humans.
01:05:01.340
Yeah. So people have looked a little bit, a little bit. Although, again, I would say even
01:05:05.240
there, the data is not definitive yet. But you're right. There have been some studies
01:05:09.100
where people have looked at the effects of metformin on exercise, both resistance training
01:05:15.460
and cardiovascular training. Yeah. I'd say the data is unclear, although there is some reason to
01:05:21.460
think that metformin might impair what people think of as the positive response to exercise. Complete
01:05:26.040
tangent. But I agree with you. The fact that that hasn't been done for rapamycin in humans is
01:05:30.940
a shame and it should be and hopefully will get done sometime in the near future. I wish I could
01:05:35.620
tell you why that's the case. I'm just sort of telling you what the observations are. My intuition
01:05:40.640
is that part of this comes down to the effects of rapamycin on chronic inflammation, which we also
01:05:48.040
know increases with aging and can impair synthesis of new muscle as well as preservation of existing
01:05:54.960
muscle. And so I think you've got some competing interests here in that rapamycin inhibition of
01:06:00.280
mTOR complex one by rapamycin. It might actually somewhat impair synthesis of new muscle, even at the
01:06:07.400
doses that seem to promote longevity in rodents. But it might actually preserve muscle because it's
01:06:12.900
having this more broad anti-inflammatory effect. And so this is why I think it's hard to get to a
01:06:19.600
specific, detailed, mechanistic answer to your question because people haven't really started
01:06:24.480
to disentangle those things. The last thing I'll mention is that I'm a little bit wary of
01:06:31.000
extrapolating too far from the rodent studies to humans in the context of sarcopenia in particular.
01:06:38.240
And in particular, I'm talking about mouse studies to humans. Mice are not, at least the commonly used
01:06:43.340
inbred mouse strains, are not particularly prone to sarcopenia with age. There are some rat models that
01:06:49.320
are better. And so I worry a little bit about the use of mouse models in particular to try to say
01:06:55.600
this is or is not going to be, have an impact on sarcopenia in humans. And I'm not talking so much
01:07:02.120
about rapamycin in this context, but I'm talking more about the studies of protein restriction and
01:07:06.160
branch chain amino acid restriction, which in mice seem to have some positive effects on longevity.
01:07:12.000
But because mice, they don't develop sarcopenia to the same extent or in the same way that people do,
01:07:17.420
I would worry a bit about extrapolating from that to say that it's going to have those same
01:07:22.400
beneficial effects in people where sarcopenia seems to be much more important for quality of
01:07:28.300
life, probably life expectancy, but certainly quality of life in older adults. So I just want
01:07:33.500
to make that caveat. We need to be a little bit careful about extrapolating from mouse studies to
01:07:38.340
humans in the context of muscle preservation, muscle function, and sarcopenia.
01:07:42.120
I think that's actually really important. It's certainly one of my gripes with people who tend
01:07:46.840
to over-index on protein restriction in animal studies, which is A, the model itself, B, the
01:07:54.780
environment in which the model exists. If you're living in a sterile environment where there aren't
01:08:01.240
curbs to step off and places to fall and injure yourself, I mean, one only need look at the mortality
01:08:08.380
data for people over the age of 75, even over the age of 65 if they fall. It's an enormous
01:08:15.000
cause of not just death, but morbidity, total destruction of quality of life.
01:08:20.460
I want to ask you both a question, or you can both chime in, and whoever has a stronger point
01:08:24.880
of view on this, maybe I'll start with you, David. Do we know from the laboratory in mice,
01:08:31.680
for example, what the tissue specificity is of rapamycin? Do we have a sense that we are getting
01:08:39.080
uniform mTOR blockade, or do we get the sense that, you know, no, it's disproportionately happening in
01:08:46.260
the liver, or it's disproportionately happening in the adipose tissue? I mean, because this would
01:08:50.500
factor into it. In a dream world, you might construct a version of a rapalog that also has
01:08:57.720
some tissue specificity in addition to what everybody's talking about, which is complex
01:09:02.200
one specificity. So, David, anything you can add on that? Matt answered your question perfectly well,
01:09:08.180
and I think it shows you the complexity of the issue. It's not only mTORC1 versus mTORC2,
01:09:12.600
it's which cell type? Is it muscle fiber? Is it inflammatory cells, immune cells? At what dose?
01:09:18.340
Is it which process? Autophagy? Is it protein synthesis? So, these are very complicated questions.
01:09:23.040
Now, on to your question, Peter. Certainly, if you dose it high enough, in our experience,
01:09:27.200
you will inhibit mTORC1 in all tissues that we looked at. It takes a little bit of time. If
01:09:31.660
you're talking about classic rapamycin to get in the brain, typically we need to do a little bit
01:09:35.380
of a loading dose, but you'll get it into the brain. Now, there's been some discrepancies. Some
01:09:39.560
people say immediately in our hands, it usually took a couple of doses to get in the brain.
01:09:44.260
Was that a couple of doses without interruption?
01:09:47.020
Yeah, typically where we did not let a trough level to get too low. This would have been probably in a
01:09:51.800
mouse maybe every eight hours or something, maybe every 12 hours. So, it's pretty aggressive.
01:09:58.560
So, your view based on those data, if you were extrapolating, is that if you were taking rapamycin
01:10:04.160
weekly, you're probably not getting CNS penetration?
01:10:08.360
Probably. With classic rapamycin. And there was some, you know, in terms of the pharma world,
01:10:13.460
people that wanted to treat tuberous sclerosis where you get these tubers in the brain,
01:10:17.600
they did not think rapamycin was adequate for that because of CNS penetration. But again,
01:10:22.800
very talented people have argued differently than that. But our experience certainly was that the
01:10:28.460
brain seemed more resistant. In fact, sometimes you would stay in the brain, you'd see almost like
01:10:32.440
a peripheral inhibition, like it kind of permeated a little bit from blood vessels in the dura and
01:10:38.000
stuff. But I think the more relevant question, Peter, is at these lower doses that people take
01:10:43.080
potentially for healthspan, lifespan studies, in the ITP studies, what are the tissues that are most
01:10:49.620
affected? Matt may know, but I don't know. And my feeling is that it's not going to be so equal
01:10:54.960
in those situations. Those are very low doses compared to what we would give to rapamycin.
01:11:01.180
My bet is that there's much more variation, and that might actually be very interesting
01:11:04.900
to know. A critical study that has been done in worms with other modulators of aging, which as far as
01:11:12.760
I know has not been done in a mammal for the mTOR pathway is, if genetically we inhibit mTOR in the
01:11:18.780
muscle, in the liver, in the brain, which one has the most prolonged lifespan, healthspan impact?
01:11:25.000
Matt, I don't think that study has been done. Not systematically. There's a little bit of data
01:11:29.420
on hypomorphic mTOR alleles, and gosh, I wish I could remember the outcomes of these. Veronica Galvin's
01:11:36.980
done some stuff for dementia, brain aging. I don't know about lifespan. I think Torrin Finkel may have
01:11:43.200
done something, but in general, it has not been done outside of, if it's been done, there was like
01:11:48.260
an adipose-specific knockdown, knockout, maybe liver-specific, but certainly not systematically
01:11:55.700
looking across different tissues. This does get at this question. It's like, okay, there's lifespan,
01:12:00.440
but then there's also the health of all the different tissues. And my bet would be that you actually
01:12:03.900
want to impact all tissues. No, I used to go to aging meetings, and I would always challenge when
01:12:08.960
at the speaker dinner, I would say, tell me a biological system that does not age. Give me
01:12:14.800
one where you don't see the impact on aging from the biochemical, cell biological to physiological
01:12:20.000
level. And as far as I know, no one has ever told me one. So I think, Peter, we need better
01:12:25.280
information under these sort of lower doses, quasi-intermittent with a feeding cycle to understand
01:12:31.380
answers your question. And Matt alluded to this before. Everyone looks at S6 kinase and its
01:12:36.440
substrate, S6, phospho-S6. There's not that much evidence it matters so much for lifespan. There's
01:12:41.920
some. What are the real relevant targets there? Let me just add on, because I think a lot of what
01:12:46.760
you said, David, is spot on and may be worth extending a little bit. So this last point about
01:12:52.040
which substrates, there's very little information about other mTORC1 substrates or mTORC2 substrates in the
01:13:00.220
context of this question of when you look across tissues, how much inhibition do you get? And it
01:13:06.820
very likely, as David already mentioned, even rapamycin doesn't affect all of the mTORC1 substrates.
01:13:13.060
And you would expect that at higher or lower doses, the relative effects on different substrates are
01:13:19.680
going to be different. So there have been a few studies looking at S6 kinase and maybe mTOR
01:13:24.340
phosphorylation of itself across tissues in the context of aging. And there are some variations, but I will
01:13:31.380
also say those studies have differed from each other because the way the experiments were done were the
01:13:36.720
mice fasted and refed before you measured mTORC activity, which affects mTORC activity, wasn't the same across
01:13:43.820
the study. So the real answer is we don't know. This brain penetration question, again, as David, I think,
01:13:49.320
correctly noted. There's disagreements out there about how effectively does rapamycin cross the
01:13:55.200
blood-brain barrier? How much rapamycin do you need to get inhibition of mTORC1 in the brain?
01:14:00.500
What I can tell you from our own studies is certainly at higher doses, and I think this matches what you've
01:14:04.880
seen, David, is that we see potent inhibition of mTORC1 in the brain after repeated dosing at higher
01:14:11.720
doses where we're using IP injection. We haven't really compared this to lower doses where the rapamycin is in
01:14:17.400
the food. The one thing I'll say is, and this is speculation, but I think it's reasonable speculation,
01:14:22.820
we know that with age there is a decline in the function of the blood-brain barrier, that many
01:14:28.320
molecules penetrate the brain better in older animals compared to younger animals. I speculate
01:14:34.160
that that's probably true with rapamycin. And so in the context of aging, it wouldn't surprise me if you
01:14:39.600
actually get better penetration of rapamycin across the blood-brain barrier in aged animals and in aged
01:14:46.500
people, potentially. But I don't know of any real data to support that. So these are all questions
01:14:52.060
that I think need answers, and there just isn't much out there right now.
01:14:56.360
A couple of questions, and then a follow-up comment. What's the size of rapamycin? How
01:15:02.580
It's almost exactly 1,000 Daltons. In the world of small molecules, it's a big small molecule.
01:15:07.960
A hydron atom is a Dalton, so it's about 1,000 hydron atoms in sort of weight. Most small molecules
01:15:18.280
What's the size at which you can easily traverse the blood-brain barrier?
01:15:22.140
I think here this is not as relevant because I think it's a very, very lipophilic molecule.
01:15:29.460
Yeah. I almost see like a lot of it gets trapped in the membrane. You almost sort of need to sort of
01:15:33.060
push it through. And you know, and the brain has a lot of things like myelin, which are all very
01:15:36.740
lipophilic. So I think there's almost like a sink of sort of trapping rapamycin in places that
01:15:41.220
maybe it's not so effective. The only anecdote I would add here, and I don't know if it means
01:15:45.960
anything. I would love to have a crystal ball that says in five years, we'll have a better answer to
01:15:51.100
this particular question. But there's a biomarker called C2N. I don't know if you guys are familiar
01:15:55.500
with it. It's a biomarker that we use clinically in humans, of course, to look for amyloid in the
01:16:02.160
serum. But it's very highly correlated with amyloid in the CNS, and it's very highly correlated
01:16:07.260
with amyloid PET scans. So obviously, in patients who are high risk for Alzheimer's disease,
01:16:14.740
if they're in a clinical trial, you might be able to justify amyloid PET or lumbar punctures to look
01:16:20.820
for amyloid in the cerebral spinal fluid. But not only does that come with the case of a lot of
01:16:26.600
radiation and potential morbidity, respectively, for those procedures, it's simply not practical
01:16:31.600
if you're clinically practicing medicine. So this C2N assay, which was approved a couple of years ago,
01:16:37.860
has become a really important part of how we manage risk in our high risk patients. And this
01:16:45.280
is very anecdotal, but for our very high risk patients who are showing amyloid already in the
01:16:51.820
plasma, I believe we have put two of them on intermittent rapamycin. So anywhere from five to
01:16:59.460
eight milligrams once a week, and in both cases, the C2N score has improved, meaning every three
01:17:07.240
months when we are checking the amyloid concentration, it's going down. There are 10 leaps of faith you'd
01:17:13.640
have to take there. Does that mean the amyloid is going down in the CNS? I don't have to spell that
01:17:19.140
out to anybody who's reasonably thoughtful. But I think your point, David, about by definition,
01:17:24.680
these patients are aging. So maybe their CNS, their blood-brain barrier is not as robust,
01:17:28.800
even though that's a very low and clearly infrequent dose of rapamycin, maybe it is making
01:17:35.180
its way into where it matters. Alternatively, it may not be making a difference where it matters,
01:17:39.500
and it may simply only be making a difference in the periphery where presumably it doesn't matter. So
01:17:43.380
there's just a lot here. Let me stop you there because I actually want to present a different
01:17:47.260
hypothesis, which is that it's actually the periphery that may matter for the brain. And there's
01:17:51.840
two lines of evidence that I can point to that might support this. One is, you know, we've worked for
01:17:56.440
many years in my lab in a mouse model of childhood mitochondrial disease called Lee syndrome. It's a
01:18:01.660
complex one deficiency in the mitochondria, but it's a brain disease. So it causes neurodegeneration
01:18:06.720
and lesions in very specific regions of the brain. So we did do an experiment along the lines of what
01:18:12.700
David was asking about in the context of longevity, which hasn't been done, where we knocked down
01:18:18.400
mTOR complex one. This was in the case of an S6 kinase knockout in different tissues. And I expected
01:18:24.820
it would be the brain specific knockout that would lead to rescue of the disease. Turns out it didn't
01:18:29.660
at all. It was the liver specific knockout that led to partial rescue of the disease. So there could be
01:18:34.940
a tissue signaling piece, and that could be metabolic. You could imagine inhibiting mTOR complex
01:18:40.120
one in the liver would lead to systemic metabolic effects. So I think that's a case in point where
01:18:45.440
you can get effects on a brain disorder. I'm not at all saying the mechanism there is the same as
01:18:50.000
neurodegeneration and aging, but you can get an effect on a brain disorder from inhibiting mTOR
01:18:55.580
complex one in the liver. The other thing that I think is super interesting, and there's
01:18:59.060
accumulating compelling data that systemic immune dysregulation drives dysfunction in many parts of
01:19:06.320
the body, including the brain. And in fact, with age concomitant with the breakdown in the blood brain
01:19:11.520
barrier, you actually may see higher penetration of peripheral activated immune cells into the brain.
01:19:17.840
And that's driving some of the inflammation in the brain. You could easily imagine, and again,
01:19:22.520
this is total speculation, but I think it's plausible that this is at least partly right.
01:19:26.420
You could easily imagine rapamycin's effects on the peripheral immune system would then reduce
01:19:33.100
the transfer of peripheral immune cells to the brain, or at least inflammation caused by those immune
01:19:40.400
cells. So it would not shock me at all if you don't really need to get high levels of rapamycin or
01:19:46.120
high levels of mTORC1 inhibition in the brain to derive some of these benefits that people have
01:19:51.680
seen, at least in laboratory animals. And our rationale for this, because of course,
01:19:56.500
someone listening to this would be understandably thinking, what the hell are these guys doing?
01:20:01.940
Why would you be giving people rapamycin when you have no idea if it works? And why would you be
01:20:08.060
doing it in somebody with elevated amounts of amyloid beta? I think part of it is just the hypothesis,
01:20:13.820
which is, look, we pretty much know that there is no meaningful treatment for this condition.
01:20:19.700
And we also know that once you've exhausted all lifestyle measures around treating people with
01:20:27.820
MCI, mild cognitive impairment, you're not going to rescue everyone. And when you understand these
01:20:33.580
potential improvements, specifically around inflammation and autophagy, we can debate the
01:20:39.180
relative importance of each of these. We didn't talk about senescent cells. Let's also come back
01:20:43.720
to that in a moment. It makes sense that this inhibition could have an effect. And I think your
01:20:48.220
point, Matt, is an excellent one that I hadn't really considered truthfully other than just through
01:20:53.640
broad reduction in inflammation. But you're right. We could be thinking about this through the lens
01:20:59.080
of less PBMC activity in the periphery should improve it. And Alzheimer's disease is a very complicated
01:21:05.900
disease with multiple pathways. There are these very lipid-dependent pathways, and there's a lipid
01:21:12.320
type of Alzheimer's disease. There's a really inflammatory type of Alzheimer's disease. I think
01:21:18.000
all of this basically speaks, hopefully screams, towards more clinical research being done.
01:21:24.640
This gets to a broader point. We've already alluded to the incredibly slow timeline for rapamycin's
01:21:32.140
transition into humans. And the net result of that was a drug that was not a profitable drug,
01:21:38.720
presumably for Pfizer, for very long. And as a result of that, there has been a relative lack
01:21:44.080
of interest in studying rapamycin and instead an interest in looking at other drugs. Let's talk about
01:21:50.580
one of them now. So Everolimus, which I believe at the time was part of the Novartis portfolio. Is that
01:21:56.140
correct? I think so, yeah. How does Everolimus differ from rapamycin before we get into talking about
01:22:01.680
one of the more important studies in humans? I don't quite remember, but it's a small
01:22:06.480
modification. I thought it was a methyl group on rapamycin. Ironically, the original patents on
01:22:11.740
rapamycin did a very poor job of covering obvious derivatives of rapamycin. I was involved in some
01:22:18.440
intellectual property cases. In the patents, they talk about rapamycins, and Wyeth, who owned those
01:22:25.200
patents, Wyeth Ayers, that Pfizer eventually bought, was trying to make the argument that that covered a lot of
01:22:30.100
these derivatives. Eventually, it was ruled that that was not the case. And so, therefore, a lot of
01:22:35.060
these so-called rapalogs, these derivatives, are actually quite simple derivatives of rapamycin that
01:22:40.160
almost many chemists would come up with. And Everolimus is one of those. Since then, there's been more
01:22:45.760
sophisticated variations, but Everolimus is, I think, Matt, a simple variant, right? I think you're
01:22:50.620
right. And again, maybe just for context, correct me if I'm wrong, David, but I think we can say that
01:22:55.740
there are these classes of what people call rapalogs, which are all going to be chemical
01:22:59.820
derivatives of rapamycin. But biochemically, they work by a pretty similar mechanism. They all bind
01:23:05.260
FKBP12, and then it's that complex that inhibits mTOR complex 1. And I think the real differences are
01:23:11.840
more around bioavailability, maybe tissue distribution, and how long the drug lasts before
01:23:18.640
it gets metabolized. I think all of these things are broken down by cytochrome P450 enzymes, and so you're
01:23:24.240
going to get differences in peak and trough levels based on the bioavailability and clearance, and
01:23:30.840
then maybe some differences in tissue distribution. But I think those are the primary things that
01:23:35.500
differentiate the rapalogs. Biochemically, I think they're all pretty similar.
01:23:40.440
I tend to view them, and certainly in cells and culture, they act identically. I think from a
01:23:44.600
biochemical point of view, at least the original rapalogs were pretty much identical. And in many cases,
01:23:50.020
frankly, I don't want this to sound pejorative, I think they were patent plays. I think they
01:23:54.200
were ways to try to get a new chemical entity that then had a longer life than rapamycin. I think
01:23:59.840
even Wyatt Ayers did that. They had a molecule, I think CCI-779, I think was its name, which was a
01:24:05.560
simple rapamycin derivative. And a lot of their cancer studies, they used that molecule instead
01:24:10.000
of rapamycin. Because as we spoke, rapamycin basically became off patent very early.
01:24:15.980
It's interesting, by the way, how expensive it still is. Even as a generic drug, it's still a
01:24:21.620
comically expensive drug. But it speaks to probably the lack of alternatives. So we alluded
01:24:26.800
to something that happened that was really remarkable, if I'm not mistaken. I think it
01:24:30.240
was even April of 2009. I kind of remember this pretty well. Fast forward five years,
01:24:36.360
five and a half years, I suppose. It's December of 2024. I didn't yet know you guys. You and I
01:24:42.660
wouldn't meet David for another year. But that was a very important day. Because I had already become
01:24:50.580
really obsessed with rapamycin, but was pretty much distraught that it would never make sense
01:24:56.940
to take as a human. That it would never go on to become a human gyroprotective agent. Because
01:25:02.220
despite how impressive all of the data were in all of these animal models, I just couldn't get out of
01:25:09.620
my mind. All those transplant patients, I was forced feeding rapamycin to, like tic-tacs and
01:25:14.720
chiclets. And I was just like, hey, this can't be a good thing if you're in the business of living
01:25:20.040
longer. And if it wasn't literally the day before Christmas, if my memory serves me correctly,
01:25:26.580
I got an embargoed copy of a paper by Joan Manick, Lloyd Clickstein, and others that seemed to at least
01:25:34.140
challenge the very foundation of that. And of course, Matt, you already made a lot of good
01:25:39.320
points about this, which is that thinking about rapamycin might have been a bit premature.
01:25:45.580
So either one of you guys, why don't you walk us through the study in the Australian senior
01:25:50.900
citizens that I think for many people was, I don't know, for me at least, a huge turning point in how
01:25:56.840
we thought about this drug. I'll say to me is that that paper, the Joan Manick paper with
01:26:01.680
rejuvenation of the immune system, I think will be seen in the aging field as certainly a milestone
01:26:06.540
paper along with the ITP paper. As far as I know, it's really the first where you actually
01:26:12.100
rejuvenate some organ system in a human being, right? And so I think her study really was mind
01:26:19.180
blowing. I think Matt can speak more to the details of it. I would agree completely with that. And we may
01:26:25.080
want to come back and touch on that because I think as people are thinking about clinical trial
01:26:30.600
endpoints for gerotherapeutics, that's a perfect case example of a functional endpoint that you can
01:26:37.120
actually do a clinical trial on for FDA approval and show improvement in function and potentially
01:26:42.400
get a drug approved as a gerotherapeutic. So I think as a conceptual advance, it's important as well.
01:26:47.520
So just to give a little bit of history, there was actually a paper, I think it was 2009 from
01:26:52.200
Penn Zheng's lab in mice that preceded the Joan Manick paper where they showed that you could treat
01:26:58.140
with rapamycin for I think six weeks in that study and rejuvenate the immune function of a mouse. And
01:27:04.320
to me, the one experiment in there that is most compelling is they have a set of mice. I think
01:27:08.680
they were 24 months of age when they started this experiment. And then they had young mice and the
01:27:13.220
mice got either a flu vaccine or no vaccine. And then they waited and then they gave them what would
01:27:20.420
be a lethal dose of influenza if they hadn't been vaccinated. In the aged mice, they either got
01:27:25.220
rapamycin for six weeks or they didn't. And so if you're a young mouse and you don't get a vaccine
01:27:30.000
and you get this dose of influenza, there's a hundred percent mortality within, I think it was
01:27:34.140
eight days. That makes sense, right? No vaccine, you're not protected against the influenza. If you're
01:27:39.700
a young mouse that got the vaccine, a hundred percent protection. So that again, makes sense. It's a
01:27:44.220
control. If you're an old mouse, no rapamycin, you get a vaccine, only 30% of the mice actually were
01:27:50.540
protected. So this is showing you the impact of just normal biological aging on the ability to
01:27:56.360
respond to a vaccine. In mice, it's about 70% of the time you don't respond to the vaccine and you die
01:28:02.680
than if you get a subsequent influenza infection. Interesting parallels to humans as we've learned over
01:28:07.900
the last four or five years. The cool thing in that study was if the mice got six weeks of rapamycin
01:28:13.200
treatment before the vaccine, they were then a hundred percent protected. These are old mice.
01:28:18.960
They're now almost 28 months old. So this is sort of an amazing demonstration of immune rejuvenation
01:28:26.040
in an aged mammal. So I think that study is what really set the stage and allowed Joan and the group
01:28:32.100
from Novartis to be able to move forward and convince the people who had to fund this study that
01:28:37.420
there was a reason to think that mTOR inhibitors might do the same thing in humans. So the design of that
01:28:42.860
human study conceptually is very similar to the mouse study I just laid out, except of course they
01:28:48.240
didn't give people lethal doses of influenza. But what they did do was they enrolled healthy older
01:28:53.420
people. I think they were over the age of 65 and there were some set of pre-existing disease that
01:28:57.640
they would be excluded for. So they were considered relatively healthy for their age and they got either
01:29:03.700
placebo. I think in the first study they tested three different doses of everolimus. So it wasn't
01:29:08.680
rapamycin, but I think we can just think about it the same as we would rapamycin based
01:29:12.840
on our earlier discussion. So there are a few interesting things here. So they got, I think,
01:29:16.880
everolimus for six weeks or a placebo and they got either... It was five milligrams once a week,
01:29:23.160
20 milligrams once a week, and I think it was one milligram daily was the third... That was what I
01:29:28.400
was going to say. So I think between the two of us were close, if not spot on. Yeah. So that was for
01:29:33.040
six weeks and then they gave a flu vaccine and then they looked at antibody titers. I don't know if it
01:29:38.700
was this study or a later one where they looked at viral gene expression as well, and then also
01:29:43.420
subsequent infections over the next, I don't know, six or 12 months or something like that, respiratory
01:29:48.400
tract infections. So the first paper, what the first paper showed was, I think, pretty convincing data
01:29:54.080
that at least at the five milligrams once a week and one milligram daily dose, there was a boost in response
01:30:02.820
to the vaccine as measured by antibody titers. So that supported the idea that similar to what had
01:30:08.620
been shown in mice, you could in fact, to some extent, rejuvenate the ability of the aged immune
01:30:13.980
system in humans to respond to a vaccine with transient dosing with a rapamycin derivative,
01:30:21.240
everolimus in this case. The other thing though, that I think was super important about that paper
01:30:25.340
was it was pretty large. I mean, not huge. It wasn't like a phase three, but there were
01:30:28.840
hundreds of people. Yeah, I think it was about 80 per arm. Okay. So hundreds of people in this study
01:30:34.540
who got everolimus who didn't have an organ transplant and weren't taking other immunosuppressants.
01:30:41.000
And the side effect profile, at least in the five megs once a week group, was essentially no
01:30:46.340
different than placebo. And so I think that study started, and it's been slow because there's still
01:30:52.120
a perception that rapamycin has a lot of bad side effects, but that started at least some people in
01:30:57.600
the community thinking, and this I think is getting what you were talking about, Peter,
01:31:01.860
maybe it is possible that lower doses of a rapalog in relatively healthy older adults could be well
01:31:09.300
tolerated. And maybe this idea that as a gerotherapeutic, we might be able to give rapamycin
01:31:14.360
to older people, maybe it's not so crazy. I think that's one of the important aspects of the study,
01:31:19.400
independent of the potential immune rejuvenating effects, which I don't want to minimize,
01:31:23.480
because that's hugely important. I actually think both of these things are important things that
01:31:27.300
that study set the stage for. And I think from that study comes a word,
01:31:31.600
certainly it wasn't coined in that study, but in my mind at least, it went from,
01:31:36.160
we shouldn't think of this as an immune suppressant, we should think of it as an immune modulator.
01:31:41.660
And that was a clear example of how you take at least an aged immune system and make it more
01:31:48.660
robust. And it might be, in fact, it very likely is the case that you can also suppress the immune
01:31:54.820
system. Interestingly, these are the same parts of the immune system. I mean, immune system,
01:32:00.120
we talk about it with one word. It's a very complicated system, but it is the same immune
01:32:04.680
system that is there to fight a virus that is also there to reject an organ. I mean, these are
01:32:09.740
not just T cells, but this is part of the cellular immune system. So that also, I think,
01:32:16.620
I was living through this at Hopkins, the age of the immunosuppressants. I mean,
01:32:20.820
remember how miraculous cyclosporine seemed and then FK506. And rapamycin, to some extent,
01:32:26.520
got caught up in being this generic sort of immunosuppressant. But the truth is,
01:32:31.340
when you looked at the data in cells and culture, it's actually not so easy to inhibit in some of those
01:32:38.000
immune activation assays and culture. Rapamycin is pretty weak. If you look at the data in mice,
01:32:43.120
it never looked like FK506 and cyclosporine, but it got caught up with that name because that was
01:32:49.700
sort of that revolution that was happening. And I think as you and Matt have said, that has sort of
01:32:54.660
persisted, but it never kind of looked. I don't think any patients are using rapamycin today
01:32:59.740
with the exception of legacy patients. In other words, I've talked to many transplant surgeons
01:33:05.320
and said, is rapamycin anywhere in your immunosuppressive regimen? And I've never heard anybody say yes.
01:33:12.660
Now, obviously, there's going to be somebody listening to this who still uses it. But I think there are
01:33:16.620
patients who still take it who received transplants 25 years ago. And it's part of their regimen and
01:33:23.060
it's working for them and no one's willing to shift it. But I think you're right. And one part of the
01:33:27.160
story I've never familiarized myself with is the literature that led to its approval for transplant
01:33:33.620
patients in 1999. You would be more familiar with that, of course, than I am.
01:33:37.460
Yeah, I don't quite remember. But I remember this study that people who take immunosuppressants
01:33:41.800
chronically have higher rates of certain types of cancer, which, of course, makes sense. Rapamycin
01:33:46.620
does not. And it was justified at the time that the reason rapamycin did not is because it itself
01:33:52.280
has anti-cancer property. Now, the alternative is that it doesn't actually impact the immune system
01:33:57.400
in the way that the other ones do to cause that. And that's never actually been quite resolved.
01:34:01.220
I think all of you are very right to say that this is not a traditional immunosuppressant in
01:34:06.740
any way. But that name has been attached to it. And people say, yeah, I don't want to get infections
01:34:12.020
by taking rapamycin. And I think there's almost no evidence that there's actually an increase in
01:34:15.600
infections at all. Let me ask you guys a question. We're going to come back to talking about broader
01:34:20.380
topics. But do you believe that if you could look at the epigenome of the T cells in those patients in
01:34:29.240
the Manick-Clickstein study, do you believe that you would see a change in the methylation pattern
01:34:35.240
pre- and post-rapamycin? Absolutely. But I think what you're really asking is, would we see a change
01:34:42.340
in the methylation pattern that is what people are calling a reversal of biological aging?
01:34:47.660
It's exactly where I'm going, which is, given our shared interest in that topic as well,
01:34:51.960
which is, is rapamycin effectively doing that? Is it rewriting the epigenome? Is it
01:34:59.180
undoing some of the aging of the T cell? And is it writing that code via methylation onto the
01:35:07.300
epigenome? I don't have a strong enough feeling to make a strong prediction there. Like I said,
01:35:12.040
there's no question you will see a change in the epigenome, but that's kind of just saying
01:35:15.460
everything big that you do to a cell is going to affect the epigenome. I'm less convinced that
01:35:20.020
these epigenetic clocks are really measuring from a biological aging perspective what some people
01:35:25.380
think they're measuring. I don't have such a strong feeling that rapamycin would reverse
01:35:30.200
what people are calling the epigenetic aging clock universally. I think in some contexts it will.
01:35:37.300
In T cells in particular, I don't know. I mean, it's a really interesting question. First of all,
01:35:41.820
what are the canonical age-related epigenetic changes in T cells and how closely are those linked to the
01:35:48.320
functional declines that we see with T cells that go along with aging? I don't think that's really
01:35:54.040
been carefully fleshed out. And so I guess I'm just less convinced what the epigenetic
01:35:59.080
clocks are actually measuring to be able to say with any level of confidence that rapamycin is
01:36:04.400
going to reverse it. No, I think the current versions of the clocks are not measuring anything
01:36:08.920
that's of interest, truthfully. But I still wonder if we just don't have the technology yet
01:36:13.200
to actually read this at CPG resolution, and therefore we don't really know what the heck is going on.
01:36:19.980
When we use these crappy microarrays to read these things, when we're sort of averaging out
01:36:25.800
methylation patterns, I think it's like trying to play the piano with mittens on. It's totally
01:36:31.320
unhelpful. But if you can take the mittens off and put your fingers on, it's a different sport.
01:36:36.600
To get to Matt's point, we had actually tried to look at the impact of rapamycin on specific
01:36:41.540
methylation patterns, not only on the DNA itself, but also on histones and using a variety of different
01:36:47.020
tools. And the truth is, we never published this because we almost found nothing specific. And all
01:36:52.120
the impacts really were from the cell cycle delay. Once you sort of normalize that away,
01:36:58.400
you couldn't say, hey, mTOR inhibitions regulating K27, this or that. There wasn't there. That signal
01:37:05.440
wasn't there. It really was an impact of delay. And so I agree with Matt, you're going to see impacts.
01:37:10.420
But why, David? So that's very interesting. But how would that explain what we just saw that in six
01:37:19.280
weeks, which is nothing in the span of a person's lifetime, six weeks of inhibiting mTOR? And again,
01:37:27.200
let's do it in the mouse experiment because that's so much more dramatic. And now admittedly, six weeks
01:37:31.340
might be analogous to a year or so in a human's life. But in a relatively short period of time,
01:37:36.880
you have a log function change in the immune system of the older mouse. It's hard for me
01:37:42.620
to understand how that could be explained by something that is just cell cycle specific and
01:37:47.660
not a fundamental rewriting of the genetic code of that cell. Again, I could be just completely
01:37:53.620
naive here, but it seems so profound. Peter, this gets to the fundamental question here is what is
01:37:59.220
wrong with the aged lymphocytes and what does rapamycin do to them to fix that? And so what I'm telling you
01:38:05.300
is that cells and culture, we always imagine there's a signal transduction pathway from mTORC1 to a
01:38:11.000
specific epigenetic change. What I can tell you is we found no evidence for that. Now that inhibition
01:38:17.460
of mTOR in a living system with lymphocytes that are impacted by many different signals coming at them
01:38:24.080
will acquire a different state that's reflected epigenetically. And I pretty much think that's what
01:38:28.560
Matt said. A cell state, Rick Young always used to say, epigenetics is the setting of the state,
01:38:36.080
not the thing that gave you that state at the beginning, right? And this is an important
01:38:39.900
distinction. So that those cells will be in a different state, but how they got to that state,
01:38:44.940
which in essence is what we're asking, we don't know. So I completely agree with you, Peter.
01:38:48.660
They're in a different state. What I am saying is that the evidence, at least in our systems,
01:38:54.100
in cells and culture of a specific signal transduction pathway, such as the one we can
01:38:59.300
define from mTORC1 to the autophagy machinery, where there's a whole relay of proteins that we
01:39:04.880
can get to the structural level, I don't know and found no evidence for one to the epigenetic state.
01:39:11.560
Let me just add a couple of thoughts here. So one is, if you think about, go back to the hallmarks of
01:39:16.340
aging, which there used to be nine, now there's 12, epigenetic changes is only one of the hallmarks of
01:39:21.200
aging. And you can find evidence in the literature that rapamycin impacts all 12 hallmarks of
01:39:25.760
aging. But the link between rapamycin and epigenetics is much weaker than some of the other
01:39:31.040
hallmarks like mitochondrial dysfunction, proteostasis, nutrient signaling. So it's not as obvious, but I
01:39:38.160
think rapamycin is going to impact epigenetic changes with aging. And this gets back to the
01:39:43.660
complexity of the downstream part, which we haven't even touched on, all the different things that mTOR
01:39:48.240
complex 1 and mTOR complex 2 regulate. Talking specifically about the immune system, though,
01:39:53.620
I think one way to think about this, and again, I'm speculating a little bit. I think, again,
01:39:57.700
there's reason to think this is at least conceptually partly the case. We know that with aging, it's not
01:40:03.380
that immune function declines globally. There is a decline in the ability of the immune system to
01:40:09.180
respond to certain challenges and hyperactivation of the immune system towards other challenges it
01:40:15.540
shouldn't respond to. That's why we get so much autoimmunity with aging or this sterile inflammation.
01:40:20.700
Just from a very simplistic conceptual perspective, you could imagine that one of the things rapamycin
01:40:25.680
is potently doing is knocking down this hyperactivation. And this is something I wanted to
01:40:32.080
mention, but we didn't talk about. In both the MANIC study and the Panjeng study, the vaccine was given
01:40:38.640
after the transient treatment with rapamycin was stopped. I would really like to know what happens if
01:40:44.300
those mice or people were continuing to receive rapamycin when they got the vaccination. But in
01:40:49.980
the context of that design, you could easily imagine six weeks of rapamycin is enough to knock down
01:40:56.280
chronic sterile inflammation to the point where you have a resetting of immune function, which then
01:41:01.900
allows the immune system to appropriately respond in a way that functionally is like a young immune
01:41:08.860
system to a vaccine. So I think you don't even have to say that this is fundamentally an
01:41:13.840
epigenetic phenomenon to account for the observation. Functionally, we can rejuvenate the ability of the
01:41:20.780
immune system to respond to a vaccine and potentially protect against a bunch of other types of
01:41:25.720
infections going forward. I also think that's how you can sort of account for the persistent effects
01:41:31.040
that we see with rapamycin treatment transiently in mice in other places like the heart or the brain or
01:41:37.660
the ovaries or the oral cavity, where we know that six to 12 weeks of treatment is enough to apparently
01:41:44.080
functionally rejuvenate those tissues and organs and that that effect persists for some period of time
01:41:49.760
going forward after you stop the treatment. Which begs a question, to cycle or not to cycle?
01:41:56.240
So Matt, you wrote or co-authored a paper that came out earlier this year that was a survey,
01:42:02.060
not an experiment, but a survey that looked at over 300 users of rapamycin. So this is a bunch of people
01:42:08.580
who are clearly using rapamycin off-label, which is a completely legal thing to do. It just means that
01:42:13.540
there is no indication for its use. And you compared them to a group of people you tried your best to
01:42:18.360
match, nearly 200 if I recall, who were hopefully as similar as possible in terms of their health
01:42:24.840
consciousness, which would be an obvious confounder, but who were not rapamycin users. Can you give us some
01:42:29.940
of the highlights of what that survey discovered? So yeah, I mean, I think you described the study
01:42:34.560
pretty well. And I think it's important to be cognizant of all of the limitations that go along with the
01:42:39.900
study like that, because it was all self-reported, all survey-based. We got in some ways lucky in the
01:42:46.340
sense that the two populations, that what we would call the users and the non-users, appear to be pretty
01:42:51.140
similar in terms of demographics and lifestyle habits. And as you said, seem to be similarly
01:42:56.580
health conscious. It's clearly a biased cohort. So if you look at the responses that the individuals
01:43:01.880
gave to the surveys, I don't have it sitting in front of me, but in terms of lifestyle factors,
01:43:06.780
this is a population that is not normal for what we would think of as middle America, much more
01:43:12.280
health conscious than I think we would see if we had a swath of just middle America. But for what it's
01:43:17.260
worth, they seem to be pretty similar. And so there were a few take-homes from that study. I think
01:43:22.100
the biggest take-home for me is that there really was no evidence when you look between the people
01:43:28.160
who were using rapamycin off-label and the people who'd never used rapamycins for significant side
01:43:34.320
effects of any sense other than mouth sores. One of the surveys was a list of, I think, 30 or 40
01:43:40.180
potentially common side effects that have been associated with rapamycin or with other drugs.
01:43:45.220
And the question was very simple. For people who'd been using rapamycin for at least three months,
01:43:49.880
have you experienced any of these in the past three months? And then for people who never used
01:43:54.220
rapamycin, same question. The only thing that came out as statistically significantly more common in
01:43:59.840
the rapamycin users was mouth sores. And that makes perfect sense. That's the most commoning side
01:44:05.040
effect that organ transplant patients experience. And lots and lots of people who've used, I think,
01:44:09.780
Peter, you've talked about your experience with mouth sores.
01:44:12.220
I have a wicked one at the base of my tongue right now that I almost burnt before this podcast.
01:44:20.120
I was just about to say, it's my only biomarker that I know that I'm getting
01:44:27.400
Right. So in a sense, it's nice to see that. And it's interesting. That was the only thing.
01:44:32.740
What's the approximate frequency? Because I think in the manic study, it was surprisingly low
01:44:40.900
Yeah, I think it was like 15% in hours as well. Yeah, I think that's exactly what it was actually.
01:44:46.160
Exactly. So 15-ish percent of people reported mouth sores.
01:44:51.600
Any idea why this is happening? Is this believed to be immune-mediated?
01:44:55.640
I don't have a good explanation. David, do you?
01:44:57.780
So I have a couple of thoughts. I think first, you're obviously not looking at the rest of your
01:45:01.420
GI tract. So you don't really know what the potential sores are elsewhere. I mean,
01:45:05.440
these are epithelia that are turning over in a couple of days. And we know from many studies,
01:45:10.060
genetic as well as pharmacological, that rapamycin tends to impact hyperproliferative cells.
01:45:15.800
If you look at, for example, the impact of mTOR hypomorphs in brain development,
01:45:20.440
it tends to be when you make the telencephalon, the cortex, where there's massive bursts of
01:45:24.400
proliferation. Lymphocytes, as we talked about, divide every eight hours. That's pretty atypical
01:45:29.500
for a mammalian cell. I would argue it's sort of epithelia proliferating fast, and you're slowing
01:45:38.440
We don't see side effects at the fingernails and the hair, which are other places where you would
01:45:43.020
expect to see it, at least based on chemotherapy traditionally.
01:45:46.760
Yeah, although there are studies arguing, for example, I know we've even done this. If you
01:45:50.620
give high-dose rapamycin before you give some chemotherapy, you can actually, for example,
01:45:54.820
prevent some of the hair loss you get in mice when you give chemotherapy. But then as soon as you
01:45:59.740
remove it, it's clear that you just arrested the cells, and then they all sort of fall out
01:46:03.380
afterwards, right? Sort of in a block. One thing, Peter, that I've always told many people in the
01:46:08.420
pharma world for the mouth sores, which I know trouble people a lot. I've never taken rapamycin,
01:46:12.880
but I know it can be pretty bad. Why don't people do FK506 mouthwashes? I don't get this. Because all
01:46:19.140
you need to do is occupy. Stuart Schreiber showed this, I don't know, ages ago. If you occupy the FKBP
01:46:26.740
of FK506, rapamycin has nothing to act on in your mouth, and you'll prevent this. Because as far as I
01:46:33.320
know, FK506 does not do this, and so you just need to occupy, or even with a benign, a rapamycin-like
01:46:40.820
molecule, all you need is an FKBP binder to sop up the binding sites that rapamycin would use.
01:46:46.880
It probably depends on the frequency with which you do it and what FK506 tastes like.
01:46:51.480
Sure, but if the mouth sores are that bad, there are rapamycin FKF6 analogs. They're completely inert.
01:46:57.020
They simply bind to FKBP, but they can't then target calcineur in the case of FK506 or mTOR in
01:47:04.160
the case of rapamycin. All I'm saying is you just need to tie up your FKBP.
01:47:11.240
Yeah, interesting experiment. And I think you're probably right, but that does make the assumption
01:47:15.520
that the mouth sores are actually caused by inhibition of mTOR in those cells inside the mouth.
01:47:21.620
And I don't think we formally know that at this point.
01:47:25.220
That would be the experiment to help elucidate that.
01:47:27.780
Or a more interesting experiment, and this is something we would love to do, is whether
01:47:31.560
rapamycin toothpaste or rapamycin mouthwash or something like that, specifically delivered
01:47:36.340
to the oral cavity, is that sufficient to get some of the benefits that we've shown in mice
01:47:40.900
from systemic rapamycin treatment on periodontal disease, gingival inflammation, bone growth around
01:47:46.580
the teeth. So that's, again, a tangent from what we were talking about, but I think super interesting
01:47:52.100
Talk to me about any of the immune stuff that you saw, because you happened to run this survey
01:47:59.580
So first, to go back to the side effects, there were other side effects that were statistically
01:48:05.320
different between the groups, but they were all the other direction, lower than people
01:48:09.160
who had been taking rapamycin. Those included things like abdominal cramps. It's harder to
01:48:14.360
really develop many hypotheses around. The ones I thought were interesting were depression
01:48:19.300
and anxiety, and there's a whole growing body of literature on the role of mTOR and inhibition
01:48:26.560
of mTOR in various types of neurocognitive behavioral aspects. And so it makes me wonder
01:48:33.280
if that actually might be real, that to some extent in some people, rapamycin could actually
01:48:39.060
have some, what in this case appear to be beneficial effects, may not always be beneficial effects
01:48:44.960
on things like depression and anxiety. So I thought that piece was interesting and certainly
01:48:49.840
worthy of further study. And I know there are some people working with rapamycin, sometimes
01:48:54.580
in the context of ketamine, for things like depression, chronic pain. So I think there's
01:48:59.780
a lot of interesting biology there that hasn't really been explored.
01:49:02.780
Can you say more about that, Matt? Because I was just about to ask you about what is ketamine
01:49:09.000
I thought it was the opposite, guys. I thought rapamycin caused depression, right? I thought
01:49:12.960
in other types of trials, rapamycin depression was one of the side effects. And certainly the
01:49:19.520
Right, because ketamine is activating mTOR in the CNS, isn't it?
01:49:23.520
That's right. The data I'm familiar with and the clinical use that I'm familiar with is
01:49:27.740
the context of rapamycin actually, in combination with ketamine, enhancing the effects of ketamine,
01:49:35.520
both in terms of magnitude and how long they last. In other words, when you combine rapamycin
01:49:41.480
with ketamine, you can sometimes go to a lower dose and reduce the frequency at which patients
01:49:46.720
are using ketamine. Although, again, I think a lot of this is not published. There are at least
01:49:52.660
a couple of studies that have showed a potentiating combination effect of rapamycin with ketamine.
01:49:57.740
I think patients with severe depression, but I don't remember for sure off the top of my head.
01:50:03.760
I've talked to psychiatrists who are using this combination who at least give anecdotal reports
01:50:09.840
of pretty potent outcomes in some patients who have severe chronic pain from combining rapamycin
01:50:16.440
with ketamine. So again, I think it's pretty early. A lot of this is being done off-label and is not
01:50:21.220
being written up the way we would like it to be reported in the literature so people can learn
01:50:26.460
from each other. But there's absolutely people using that combination now in clinical practice.
01:50:32.300
That's interesting because I think the initial, I think it was from Dumont at Yale,
01:50:36.160
I think the original ketamine study argued that rapamycin blocked the effect of ketamine.
01:50:40.760
And that was partly the argument that mTOR was involved. I think I recall also, Matt,
01:50:45.140
where you're saying that there's some discrepancy there. And it might be blood-brain barrier access.
01:50:49.480
It might be things like this that are quite different and very dose-dependent.
01:50:52.560
Sounds like we need to go back to that original study and make sure we all
01:50:55.680
we're all on the same page. So all I can tell you is I know from conversations with people who are
01:51:00.900
actually using this now that there are people using the combination of rapamycin with ketamine
01:51:05.660
and at least anecdotally sometimes reporting pretty significant changes in outcomes.
01:51:11.860
And that ketamine is intranasal, intravenous, intramuscular, does it matter?
01:51:19.560
Let's go back to the survey. The other thing that I remember jumping out at me was,
01:51:24.340
and again, lots of confounders here. If you have a healthier population who's more health-conscious
01:51:28.220
and that's why they're taking RAPA because they're literally at the periphery of what one would do,
01:51:33.200
that could easily explain the observation that they got COVID less and when they got it,
01:51:40.900
Yeah. So let me tell you what we observed in the data with all the caveats that there are around the
01:51:45.400
way the study was designed and carried out. So within, again, two populations, people who had
01:51:51.120
ever used rapamycin, they're all in the rapamycin user group. People who had never used rapamycin,
01:51:56.160
they're in the non-user group. But when you look within the rapamycin user group, we actually had
01:52:00.940
three categories of people in the context of COVID-19 infection. Some people didn't start taking
01:52:07.260
rapamycin until after they had had their COVID-19 infection. Some people took it before,
01:52:12.800
but not after or not during. And then there were people who took it continuously throughout.
01:52:18.820
And so we tried to group them that way and look at if there were any differences between the groups.
01:52:23.160
So first of all, no difference in frequency of infection that was significant. So there's no
01:52:29.200
reason to believe based on our data that rapamycin impacted the likelihood that somebody would
01:52:34.520
get a positive COVID-19 result. This is self-reported. So we asked people to confirm that this was a
01:52:42.020
positive result from a test, but we're going by what they told us. We don't have any laboratory
01:52:46.320
confirmation. So the interesting thing was that the people who took rapamycin after they got their
01:52:53.560
COVID-19 infection looked just like the people who never took rapamycin. That makes sense. They
01:52:57.580
shouldn't. And we were looking at two things. Severity of infection, again, self-reported as mild,
01:53:03.780
moderate, or severe. And we had specific criteria for length of symptoms and hospitalization for each of
01:53:08.960
those groups. And then self-reported long COVID, as in experiencing ongoing symptoms of COVID after
01:53:16.420
a three-month period. So no difference between people who started taking rapamycin after their
01:53:22.500
infection and non-users. No difference between people who took rapamycin before their infection,
01:53:27.500
but stopped taking it. Big difference, at least statistically significant, between people who
01:53:32.420
took rapamycin throughout and all of the other groups, where people who took rapamycin throughout
01:53:38.700
had lower severity of infection. And the numbers were really small, so I don't want to make too much
01:53:45.040
of it, but statistically significantly less likelihood of reporting symptoms associated with long COVID. So
01:53:51.820
it's at least, I think, suggestive of the idea that rapamycin continuous use throughout the period of
01:53:59.120
infection and resolution of symptoms. It may be associated with a lower likelihood of severity
01:54:05.840
of outcome and lower likelihood of long COVID. And again, I think that might make sense in the
01:54:11.080
context of at least how, at a crude level, we think long COVID in particular is working and severe COVID
01:54:17.480
infections, which is there's this hyper-inflammatory or chronic inflammatory response. It kind of makes sense
01:54:23.500
that rapamycin use may have benefits in the context of that prolonged inflammation or hyper-inflammatory
01:54:31.460
response. So that might explain what we saw in the data. But again, I think it's just suggestive and
01:54:37.060
worthy of potentially future work to really disentangle. And I will say, I don't think there's
01:54:43.540
any reason to think this is specific to COVID-19. This may be a general property of rapamycin for a bunch
01:54:50.460
of different types of at least viral infections. David, you mentioned a moment ago, you've never
01:54:55.660
taken rapamycin. Obviously, Matt and I have. Say a little bit more about that. Obviously, you're one
01:55:01.600
of the most knowledgeable people on this topic. I think it is perhaps somewhat telling and maybe
01:55:06.720
important for folks who are out there considering it to understand why your decision has been not to
01:55:11.340
take it. I always used to joke that when I was purifying amtory, I got a huge dosing. And given that
01:55:16.100
early exposure is better, I got the benefit then. I never wore gloves and it's a powder. I remember
01:55:21.580
it would get into my nose and stuff. So I've snorted rapamycin inadvertently. So I did get a
01:55:27.400
dose at the time. Now, you know, Peter, it isn't such a willful thing. It's more that it takes some
01:55:32.700
effort to go and actually do it. But I do wonder, and you and I have had this discussion, if you eat
01:55:38.600
okay and you do exercise, if rapamycin is a mimetic to some extent of a healthy diet, I know
01:55:46.320
it's more complicated than that. But if we call it that, are you getting that extra benefit, right,
01:55:51.340
at the doses in particular that we're talking about? And so that would be my biggest question.
01:55:55.980
It wouldn't be, am I afraid of it? I'm not. But will it actually do anything?
01:56:01.900
But isn't there sort of a hedging or a Pascal's wager, which is as long as you could convince
01:56:05.740
yourself that it's not harmful with the worst thing you're doing is wasting a lot of money
01:56:11.420
because it ain't cheap. So I agree. But then that's where the laziness factor comes in and
01:56:15.820
sort of figuring out how to do it and stuff. But what I would really like to know, and this is what
01:56:20.140
I'd like to study in the future, is getting back to, I think, Matt, you've mentioned it. Peter,
01:56:24.380
you have this cyclical nature, right? I'm much more interested in sort of a, because what can't I do?
01:56:29.740
If I starve myself, what happens? My body synthesizes certain nutrients,
01:56:34.800
it's breakdowns, other things to release them. And in fact, when you look at the metabolic state
01:56:39.340
of a mouse that you've starved, the levels in the blood are pretty similar. So I can't,
01:56:46.240
through dietary interventions, starve a cell of nutrients like I can in a dish. I can't.
01:56:51.720
The body fights that. And of course, eventually you run out of stores and you die, but in a normal
01:56:56.440
type of starvation situation. So what I'm much more curious about is, can I use rapamycin or other
01:57:02.760
mTOR modulators, perhaps, God forbid, even catalytic inhibitors, to take that system to a
01:57:09.200
state that I cannot simply do with a dietary intervention whatsoever? And obviously that is
01:57:14.580
not sustainable in any chronic way. We know that. If you give a catalytic inhibitor to a mouse,
01:57:19.040
you can actually kill a mouse fairly easily. It's actually hard to kill a mouse with rapamycin.
01:57:23.680
Can you remind folks again, the difference between an allosteric and a catalytic inhibitor and
01:57:27.380
what that actually is doing in the case of mTOR? So the allosteric inhibitor, rapamycin and
01:57:32.360
derivatives is going to do this partial inhibition of mTORC1. The rock that partially obstructs the cave
01:57:38.160
entrance. Exactly. The partial rock and also partially inhibit mTORC2. And there's going to be
01:57:43.460
perhaps some tissue specificity, some kinetic differences. A catalytic inhibitor, which is
01:57:49.280
basically a molecule that will compete with ATP, which is what mTOR uses to do all its business,
01:57:55.960
that will obliterate mTORC1 and mTORC2 activity. Certainly when given at the right doses. And in
01:58:02.160
our hands is highly toxic to cells and to organisms. Again, we have misdosed by mistake catalytic
01:58:10.260
inhibitors in a mouse and a mouse will drop dead. When you say drop dead, are you talking about the
01:58:14.980
same way where mitochondrial inhibitors like cyanide, which immediately cease respiration,
01:58:20.280
will kill an animal within seconds? No, it'll take usually a couple hours. The mouse will stop moving.
01:58:25.400
It'll get cold. Sometimes it'll have seizures, but it will die. But still profoundly and acutely toxic.
01:58:32.220
Profoundly bad. Yes. Which rapamycin does not do. So clearly one has to be careful of those
01:58:37.480
molecules. And the clinical experience has suggested that, right? These were molecules that were initially
01:58:41.980
thought to be potentially good anti-cancer agents. We made some of the first ones and also were touting
01:58:47.320
it from that. But I think the experience has been that they have lots of side effects. But I've always
01:58:51.980
wondered, can those molecules in a careful way be done to very much impact this system, massively
01:58:59.040
activate autophagy, massively rewire this system, maybe have epigenetic impacts very short and then
01:59:06.020
come off of that. I'm much more curious about that type of study and potential use because I feel that,
01:59:13.240
again, with diet, you can get close to rapamycin's impact. Again, this is my personal belief with some
01:59:20.380
data to support it. But what I know you can't get close to with diet is what a catalytic inhibitor can
01:59:25.600
do. I think you said that, and I've tried to make this point before, and I think you said it in a way
01:59:30.620
that I've never thought about it, or at least I've never said out loud, which is important. The point is
01:59:35.760
that rapamycin is very different than dietary restriction. They're overlapping, but they have lots of
01:59:40.880
differences. And I think you're right. You can't have the same impact on mTOR systemically in tissues
01:59:46.940
with dietary restriction that you have with rapamycin. The other side of that, though, that's
01:59:52.540
equally important maybe is that dietary restriction does a bunch of other stuff that rapamycin doesn't
01:59:57.640
do. And the potential benefits and negative consequences of all of that other stuff, I think,
02:00:03.480
are often not weighed into the equation when people are thinking about diet and comparing it to
02:00:10.200
rapamycin. The catalytic inhibitors, though, the point I wanted to make is that there's two. One is
02:00:15.780
most of these catalytic inhibitors are less specific for mTOR than rapamycin, meaning many of them
02:00:21.540
affect other kinases. Not all of them, but many of them do. And there's this whole class of what
02:00:26.200
people call dual kinase inhibitors that hit other kinases. David's shaking his head, so you can tell me
02:00:31.220
why I'm wrong. But there are other proteins that some of these molecules that inhibit mTOR will also
02:00:37.280
inhibit. And RTB-101, which we didn't talk about the subsequent studies from Joan at Novartis and then
02:00:46.380
when she went on to Restore Bio, there's this other molecule, RTB-101, that I think would fall into
02:00:53.680
these ATP competitive mTOR inhibitor class, but it also inhibits other kinases. So the specificity for
02:01:00.180
some of these molecules is less. I don't know if we know, in terms of the side effect profile,
02:01:07.680
how much of that is due to mTOR, mTORC1, mTORC2, or other kinases that these molecules inhibit.
02:01:15.180
But I do think it is worth saying, at least in the studies that Joan did at Restore Bio,
02:01:19.880
they did dose people with RTB-101 and did not see significant side effects. So you can ask whether
02:01:26.860
they saw significant efficacy, that trial actually was shut down. But it is possible, at least for
02:01:31.660
that molecule, to use it clinically at doses where there's some reason to believe there might
02:01:36.940
be some efficacy. Before letting David chime in, can I just ask a question to clarify that
02:01:41.800
in the RTB-101 trial, didn't they combine it with another agent? They did, with everolimus. So they had
02:01:48.380
two arms. One was the combination and one was RTB-101 alone, yeah. My shaking was that I was agreeing
02:01:55.000
with you. And that study that Manik did after, I was confounded by that study and perplexed because
02:02:00.720
this RTP, which they renamed, I think it was NDP-103, which was a Novartis molecule that's a
02:02:07.580
dual mTOR PI3 kinase inhibitor and actually a very dirty molecule. I remember being on some advisory
02:02:12.360
panels for Novartis and really not understanding why this molecule even existed. So you're right,
02:02:18.140
the ATP-competitive inhibitors are dirtier than rapamycin by far, but not all of them. In fact,
02:02:24.300
Wyeth had made a compound under the guidance of Bob Abraham, who's one of the pioneers in
02:02:29.440
mTOR biology, which is exquisitively specific. You can dial out PI3 kinase activity of the
02:02:35.740
catalytic inhibitors, but the kinase that was very hard to not also hit was DNA-PK, a kinase involved
02:02:42.360
in the DNA damage response. The molecule we made, Torrin-1, we never managed to dial out DNA-PK.
02:02:47.560
He did. So this Wyeth compound is a beautiful molecule. When Pfizer bought Wyeth, they de-emphasized
02:02:54.720
it in favor of dual activity inhibitors, which again, I did not agree with. I do think there
02:03:01.860
are some quite good molecules and that's the molecules that we use, these very hyper-specific
02:03:06.700
ones, and they are bad news for an animal. This gets back to low-hanging fruit that hasn't been
02:03:13.920
studied. I would love to see somebody take a panel of all of the known mTOR inhibitors in these
02:03:20.540
different classes and just ask the question, if you look in an animal model, what's the relative
02:03:26.060
benefit and side effect profile look like in the context of longevity? I'm confident that at least
02:03:33.060
in worms, you will find things that work better than rapamycin because we've already done it.
02:03:37.140
I don't know about in mice, but it seems like a really important question to understand
02:03:42.280
the biology of these mTOR inhibitors in the context of aging to know, is rapamycin really best in class
02:03:49.300
or is it just the one that we've studied the most? And that seems like a completely unknown to me at
02:03:55.100
this point. You would just have to guess that it's not best in class in the same way that the first of
02:04:00.240
anything, it could always be perfected, right? I mean, that would be your guess.
02:04:04.260
Yeah, absolutely. That would definitely be my guess.
02:04:06.720
It would be my guess too, but the balance between full mTORC1 inhibition, total mTORC2 inhibition,
02:04:12.460
I don't know the answer to that. And one of the reasons I think this hasn't been done is that the
02:04:16.220
catalytic inhibitors are actually very challenging to use. They're very hydrophobic molecules because
02:04:20.780
the catalytic site of mTOR is like a very hydrophobic site. So everyone who independently made
02:04:26.040
these molecules ended up with very greasy molecules that are not easy to dose in a mouse,
02:04:31.480
very hard to dose. You got to put them in detergents, all these things that the mice don't
02:04:35.300
like either. But I completely agree. But I would do that study, Matt, in an intermittent way.
02:04:42.140
That's the way that I would want to do that, to sort of mimic a really strong inhibition of this
02:04:51.520
Guys, why do you think that they put forward RTB-101? I mean, you made a point a minute ago,
02:04:57.240
David. I mean, it was probably more of a PI3 kinase inhibitor.
02:05:02.420
Exactly. Like, I also was confused. And the problem is when that second study came out
02:05:09.140
and it was a null study, it somehow got interpreted as, oh, wait, Everolimus doesn't work, which,
02:05:17.320
again, there's no scenario under which I would make that interpretation. But help me understand
02:05:21.140
that. Because you wrote, if I recall, Matt, you wrote an editorial on this, if I'm remembering
02:05:26.360
Right. So there were actually three studies. The study where RTB-101 was used alone was actually
02:05:32.020
the third, and that was their pivotal clinical trial. There was a second phase two in between
02:05:36.880
the 2014 paper and the pivotal where they used a combination of Everolimus with RTB-101. And I wasn't
02:05:45.760
in the room, so I don't know exactly what went into the thought process of why use RTB-101.
02:05:52.080
I've been told there are probably at least two factors that played in. One was that in cell
02:05:57.520
culture models, there was some data that RTB-101 induced antiviral gene expression. So there was
02:06:03.420
some somewhat plausible biological rationale for the endpoint that they were going after, which was,
02:06:10.380
if I remember correctly, at least for the pivotal, it wasn't so much vaccine response,
02:06:14.100
it was subsequent infections. And so the thought was, if you can both boost vaccine response and
02:06:19.720
enhance resistance to subsequent infections, that might be a combination that was useful.
02:06:26.420
So in the second phase two, the RTB-101 showed a signal. RTB-101 plus Everolimus also showed a signal,
02:06:35.360
but RTB-101 alone showed a signal. So the decision was made to go to the pivotal with RTB-101 alone.
02:06:42.740
I don't know the rationale for that. You could speculate it might have something to do with
02:06:46.360
patent life, right? And IP around longer patent life on RTB-101, clearer path to market. I don't
02:06:52.760
know for sure, but that's what happened. So there was no Everolimus in the pivotal phase three. There
02:06:58.560
are a couple of things about that trial that are worth just mentioning. One is that Everolimus wasn't
02:07:03.220
in there. So the failure of that trial absolutely should not be interpreted as a failure of rapamycin or
02:07:08.980
rapologues because there was no rapologue in that trial. The other piece though, that I think is
02:07:13.240
worth mentioning is that trial was only half completed. And the decision was made halfway
02:07:18.760
through to stop the trial because they were not hitting their FDA mandated endpoint, which was
02:07:25.020
patient reported infections, not laboratory confirmed, patient reported. So they were not
02:07:31.020
hitting that endpoint. And the decision was made to stop the trial halfway through. That was actually
02:07:35.980
November of 2019. I remember I was at a conference with Joan, the Gerontological Society of America
02:07:41.300
conference, when that news came down. I was upset. I'm sure Joan was even more upset. But if you think
02:07:47.640
about where the world was five months later, they might've made a different decision at that point
02:07:52.440
with a drug that could potentially affect vaccine response and subsequent viral infections. Regardless,
02:07:57.540
that's all history. But now Joan did go back and do a subsequent analysis on the data from that
02:08:03.960
half completed phase three. And in fact, in those patients who got the RTB 101, there was a
02:08:11.480
significantly lower risk of subsequent infection for certain viruses, among them influenza viruses and
02:08:19.020
coronaviruses. Not COVID-19 because we didn't know about COVID-19 when this was happening, but
02:08:24.160
coronaviruses as a class. The people who'd gotten RTB 101 showed a significantly lower likelihood of a
02:08:31.600
future laboratory-confirmed viral infection. So whether that trial was actually a failure,
02:08:38.000
it was a failure in the sense that they didn't get to FDA approval and they shut it down early.
02:08:43.120
Whether it was actually a failure of the drug, I think still remains TBD, which is interesting
02:08:47.840
because this wasn't a rapamycin. It was one of these ATP-competitive mTOR inhibitors. But I think
02:08:53.100
it's still a little bit unclear if the drug itself actually failed to have an impact on immune
02:08:59.200
function in the population where it was tested. But it was a very dirty catalytic inhibitor. It
02:09:04.880
impacts multiple PI3 kinases. Yeah, absolutely. And I mean, that makes it harder from the perspective
02:09:10.460
of even if it did have an impact, how is it working? Is it really through mTOR? Is it through some of
02:09:15.220
these other kinases? Is it a combination? We don't really know because it is dirty. So I always worried
02:09:19.980
that the change in sort of use of molecules reflected that that original study maybe had some issues that
02:09:25.820
were not aware of. That first study that we talked about as a milestone study was so amazing that why
02:09:32.240
wouldn't you have expanded upon that? I never understood this, but I think what you said makes
02:09:36.040
a lot of sense, Matt. Yeah. And I don't remember whenever Alimus came off patent, but it's been a
02:09:40.800
few years now. So the patent clock was ticking. I would speculate that it had something to do with
02:09:46.200
the decision. I don't think that's a skeptical point of view. That would be my Occam's razor
02:09:51.680
answer to that question, for sure. But there are now so many rapamycin derivatives. I still imagine
02:09:58.800
you could have picked one up. I'd have to, I guess, go through a lot of preclinical studies and things.
02:10:03.460
David, you've talked a lot about the impact of mTOR inhibition. You've already talked about autophagy.
02:10:11.140
We've talked about a reduction of inflammation. We haven't talked a lot about the tamping down of
02:10:15.620
senescent cells and potentially the reduction of the soluble or secretory factors. We have an impact
02:10:21.680
on proteomics. I mean, lots of things are impacted. You tend to think, if I'm not mistaken, that the
02:10:28.380
impact on autophagy is the one that might be most responsible for the life property, the altering
02:10:37.000
benefits we see of that. You want to expand on that a little bit? And Matt, I'm kind of curious to
02:10:41.380
hear your point of view on that as well. What do you think, which pathways, plural, would you rank
02:10:47.020
order as the ones that are driving this? And the reason I'm asking this, I'll tell you where I'm
02:10:50.480
going with the question in advance. It comes down to biomarkers, a topic that the three of us have
02:10:55.880
endlessly, endlessly talked about, which is, if we believe this is dominated by autophagy,
02:11:03.220
then we need biomarkers for autophagy. If we believe this is dominated by inflammation,
02:11:08.980
then we need better biomarkers for inflammation. So with that said, I'd like to hear your thoughts.
02:11:14.800
When you think of things downstream of mTOR, right, you can do a PubMed search and find mTOR and
02:11:19.760
rapamycin literally connected to anything you want. Why is that? Either there's a specific
02:11:25.520
signaling pathway to that process, or there's a simpler explanation, which to me is that mTOR is a
02:11:31.760
major regulator of protein synthesis. And if you inhibit mTOR enough, particularly if a catalytic
02:11:37.300
inhibitor, you inhibit protein synthesis, so you will impact everything. And so to me, there is
02:11:41.480
the class of downstream molecules that are impacted simply by impacting protein synthesis.
02:11:48.180
I put those in a very sort of broad category that I don't know how to study them or think about them
02:11:55.180
in any kind of specific way. There are then a whole series of processes in which there are truly
02:12:02.000
molecular connections, direct specific molecular connections, that mTOR regulates. And as you said,
02:12:07.300
Peter, autophagy, the self-eating of cellular components and destruction in the lysosome that
02:12:13.620
came up earlier, where we know that pathway. We know how it regulates protein synthesis. We know
02:12:20.140
how it regulates transcription factors like TFEB. So one that I think, if you had to put in the
02:12:25.460
molecular target of mTOR that's emerged in the last 10, 15 years as very interesting and prominent,
02:12:31.960
it would be TFEB. It's a transcription factor that what it does is promote the production of
02:12:38.120
these lysosomes, these recycling organelles. And so, yes, Peter, I would put autophagy. If I had to
02:12:45.040
pick one process that is prominently regulated by mTOR and probably accounts for some of its health
02:12:51.360
benefits, I would put autophagy. Part of that is based on a worm study that I'm sure Matt knows better
02:12:56.740
than I do, where they actually tried to look at that. They did mTOR inhibition and then they looked
02:13:01.980
at downstream pathways genetically and found the biggest impact of perturbing autophagy. Part of it
02:13:08.300
is based on common sense. It breaks down old things and allows their rejuvenation. The counter,
02:13:15.060
though, to the statement that I just made is that I'm always asked, why does mTOR impact aging and why do
02:13:21.760
other things not? And what I always say is that if you make the analogy of an old house,
02:13:28.160
you can't prevent the aging of an old house or much less rejuvenate an old house by having a plumber,
02:13:33.880
having an electrician. You need a general contractor that brings in all those people because an old house
02:13:39.640
has everything wrong with it, as we know, or an old car has every part wrong with it.
02:13:44.240
And so, I think to some extent, we almost can't ask the question, what is important downstream of mTOR?
02:13:49.240
Because the answer is mTOR is special because it does a lot of things and therefore we can't find
02:13:55.620
one thing that replicates mTOR. Otherwise, we would have already found those things. And so, I guess,
02:14:00.700
Peter, if you had to pick, I'd say autophagy is the major one. But I think the real answer as to why mTOR
02:14:06.900
and thus rapamycin are special is that mTOR does a lot of stuff. And to impact the aging process, you have
02:14:13.100
to do a lot of stuff. And this is why it goes back to that question that I always ask the real aging
02:14:17.360
researchers, tell me one thing in a cell that's not broken with aging. And the answer is, there
02:14:22.920
isn't one thing that's not broken. And so, therefore, to fix or prevent that, you have to act on many
02:14:30.020
What about you, Matt? Where do you end up on this question?
02:14:33.000
I don't disagree with anything that David said. And I think the house analogy is that's a nice way
02:14:38.160
to think of it because it is the case that mTOR globally regulates a lot of different things.
02:14:44.220
And it's probably multiple downstream processes that play a role. And I think what I would say,
02:14:49.820
though, is that autophagy being an important, maybe the most important single downstream directly
02:14:56.280
regulated mTOR process for a bunch of different, broadly speaking, aging effects is not inconsistent
02:15:03.080
with the idea that in a mammal or in a person, the anti-inflammatory effects account for many,
02:15:10.880
maybe most of the functional benefits that we see when we treat an old organism, old animal.
02:15:17.320
I think both of those things can be true. And it's probably the case that the specific effects
02:15:22.700
of mTOR may be different in different contexts, different tissues, different pathologies. So,
02:15:28.140
for example, in hypertrophy, the effects of mTOR on cell size may be most important.
02:15:34.860
In cancers, the effects of mTOR on the cell cycle may be most important. Those are tied into autophagy.
02:15:39.840
I mean, I don't know that we're going to be successful trying to point to one thing and say,
02:15:44.220
that's the most important thing. David's absolutely right, though, that in C. elegans,
02:15:48.200
at least, it's interesting because it seems to be the case that most, if not all of the benefits
02:15:53.280
of inhibiting mTOR can be directly attributed to activation of autophagy. But you go to yeast,
02:15:59.180
and it seems to be mostly the effects on global mRNA translation. So again, that may fit with the idea
02:16:05.360
that context is important here for which of these downstream processes are weighted in a relative
02:16:11.360
sense, most importantly, for the effects that we see in the context of aging. So that's kind of the
02:16:16.760
way I think about it. But again, I think you and I have talked about this before, Peter. I have very
02:16:20.840
much in the last five to 10 years shifted my thinking, particularly in the context of people
02:16:26.680
and probably in other mammals towards the anti-inflammatory effects, and particularly
02:16:32.080
the ability of rapamycin to knock down sterile inflammation in the context of an aged animal.
02:16:38.480
That seems to me that a lot of the benefits that we see in terms of organ and tissue function
02:16:43.780
can be plausibly traced back to that effect of rapamycin.
02:16:48.380
Very interesting. I mean, I think that would lead us to think that, boy, if we really wanted to get
02:16:53.640
a better handle on dosing, we would want to look deeper into biomarkers of inflammation. And we do
02:16:59.580
have more that we can look at there. I mean, you know, everybody gets their C-reactive protein checked,
02:17:04.480
but we could be looking at a whole suite of interleukins and other cytokines. But when it comes
02:17:09.940
to autophagy, boy, we've got a whole lot of nothing. Probably been three years now since I had a really
02:17:15.560
interesting discussion with Eileen White about this, who's one of the world's experts on this.
02:17:20.080
I don't think I got any argument out of Eileen that we really need a biomarker here, because
02:17:25.420
outside of the lab, when you can afford to take tissue, we don't have much going on.
02:17:30.660
I want to pivot for a sec, and we've done this before, Matt, but again, I think there are people
02:17:34.020
listening to this who maybe haven't heard it. Can you tell us a little bit about what we've learned
02:17:37.820
in rapamycin as we've pivoted to companion animals? So when we talk specifically about cats and dogs,
02:17:43.300
what is it about cats and dogs that are interesting? Well, first of all, they're a heck of a lot closer
02:17:47.880
to humans than mice are, but they're also not genetically inbred the way mice are. They live
02:17:54.180
in our environment, not a sterile environment. They consume food that probably looks a little bit more
02:18:01.440
like the food we would consume, at least in some cases. So tell us about what you've learned in this
02:18:07.020
study, which has really occupied more than a decade of your research.
02:18:11.760
So there's two other things I would add about companion animals and dogs in particular,
02:18:17.060
where most of my work has been, but this is also true for cats. One is they age more rapidly than
02:18:22.640
people do. So that's super important because that means we can actually measure outcomes of interest
02:18:28.520
in the timeframe that's compatible with a clinical trial. Secondly, they matter. More than 50% of people
02:18:35.340
say that their pet is part of their family. So there's sort of an intrinsic value, I believe,
02:18:40.700
in developing therapies that can improve health span and longevity of companion animals from that
02:18:45.900
perspective. So just to make sure, yeah, what you're basically saying is even if we learned nothing about
02:18:51.660
the longevity of humans, this would be a worthwhile pursuit in the way nobody actually cares how long
02:18:59.240
That's exactly right. And I would also say it's ridiculous to think we're going to learn nothing
02:19:03.620
about the biology of aging in humans from studying companion animals. But yes, even if we say that,
02:19:09.000
there's still value in doing these kinds of studies and improving the quality and quantity of life for
02:19:13.880
pets. So I've been involved, as you know, for a while now with a project called the Dog Aging
02:19:18.800
Project, which Daniel Promislow, Kate Creevey, and myself started, depending on how you want to do the
02:19:24.120
math somewhere between 7 and 12 years ago, with the idea, you know, sort of around what we've already
02:19:29.880
discussed, that there's a good rationale for companion dogs, pet dogs in particular, as a model
02:19:37.080
for the biology of aging, but also to be able to assess rapamycin specifically for its impact on
02:19:45.020
lifespan and healthspan metrics, because we can actually design a clinical trial. And this is a real
02:19:51.160
clinical trial, double-blind, randomized, placebo-controlled, veterinary clinical trial to answer
02:19:56.580
the question, does rapamycin, slow aging, increased lifespan, improve multiple healthspan metrics in a
02:20:03.520
reasonable time frame? So we set out to design such a clinical trial. We call it the Test of Rapamycin in
02:20:09.980
Aging Dogs. We've done two shorter-term pilot trials, also double-blind, placebo-controlled, to establish
02:20:17.040
safety, to kind of work out dosing, and then started the larger clinical trial, TRIAD, a few years ago,
02:20:24.100
which unfortunately coincided with the beginning of COVID-19. So that was challenging, but we
02:20:29.060
continued to work through that and are making progress. And so this is a trial that will
02:20:33.660
ultimately enroll 580 dogs, half get placebo, half get rapamycin. The treatment period is three years.
02:20:41.380
We're looking at multiple measures of healthspan, including cardiac function, neurological function,
02:20:47.640
activity, cognitive function. There's a few others. But I think most importantly, lifespan is the primary
02:20:53.560
endpoint. So with that cohort size, that length of treatment, we are powered to detect a 9% change
02:21:01.980
in lifespan. Is that remaining lifespan or total lifespan? That's total lifespan. So life expectancy,
02:21:09.920
it's a bigger number for remaining life expectancy. The reason why we settled on that 9%, as you know,
02:21:14.920
Peter, because you were instrumental in getting us to that point by helping to line up a group of donors
02:21:19.760
who increased the size of the study, the reason why we aligned on that percentage is because that's
02:21:25.740
towards the lower end of what's been reported in mice. And that's, in fact, what was seen in that
02:21:30.660
2009 study we talked about earlier, starting treatment in middle age in mice. So again, it's a big
02:21:36.880
question. It's unanswered. Even if rapamycin extends lifespan in dogs and in people, will the magnitude
02:21:43.180
of effect translate? That's a different question we don't know the answer to. But it makes sense to
02:21:47.880
start in the right ballpark in terms of what we think might be a reasonable thing to expect for
02:21:53.580
longevity. So that's why we went with that cohort size. A couple of other things that are maybe worth
02:21:58.480
just mentioning is that the dogs have to be at least seven years old at the time of randomization,
02:22:03.360
and they can't be sick. They can't have any significant pre-existing age-related disease.
02:22:08.840
And that's important because the vast majority of clinical trials that are done today, whether it's
02:22:13.860
in companion animals or people, are disease-specific clinical trials in patients who already have a
02:22:18.960
pre-existing disorder. This is a study of normative aging. And so we felt it was important to start with
02:22:24.920
a population that was at least age-appropriate in terms of health status. And so that's the study.
02:22:30.860
Dogs are still actively being enrolled. Any size limitations, Matt?
02:22:34.900
Yeah. So the dogs have to be between 40 and 110 pounds. And that's for the simple reason that big
02:22:40.300
dogs age faster than small dogs. So again, in order to get the statistical power that we needed,
02:22:46.060
we are working in a population of dogs that are more rapidly aging than a smaller weight,
02:22:52.080
body-size population. Can I say one quick thing? You know, you always ask me if I take rapamycin,
02:22:57.100
and my friends ask me whether they should take rapamycin because they know you and you take
02:23:01.620
rapamycin. And I always say, well, when Matt Caberlin's dog study reads out, if it's positive,
02:23:07.740
It's funny you say that, David. I say that to a lot of our patients as well. I say, look, again,
02:23:12.860
I have a relatively strong conviction. It's modestly held. It will be a lot more of a strong conviction
02:23:20.720
one way or the other, and I'll tighten my grip on it in 2026, which is about the time when we'll
02:23:26.380
have the readout of this study. So yeah, I think a lot of people, Matt, are looking to this study
02:23:31.400
potentially along with the work of Adam Solomon. Maybe we can just touch on that really, really
02:23:36.020
briefly as well as another model. Let me make a comment on that though. So even though we're
02:23:40.560
powered for lifespan, that's our primary endpoint, I'm honestly not sure that's the most important
02:23:45.040
endpoint for evaluating potential efficacy of rapamycin in dogs or people. I mean, I think
02:23:51.640
we want to think about this more broadly speaking in the sense that there may be some healthspan
02:23:57.840
metrics that are particularly and potently positively impacted by rapamycin.
02:24:04.440
I think people just also want to make sure there's no negative lifespan though.
02:24:08.140
Oh, absolutely. I agree. I would be shocked. I mean, again, we'll wait till the study's done.
02:24:12.440
I would be shocked if we see a shortening of lifespan from rapamycin treatment. Just given
02:24:17.820
everything that I know to this point in mice and the data we've gotten so far in dogs, it is possible.
02:24:22.580
And I totally understand that reasoning would surprise the heck out of me if we see any
02:24:28.640
lifespan shortening. Not to say that there aren't side effects from rapamycin, but I don't think
02:24:33.160
there's any reason to believe that it's going to have a negative impact on mortality.
02:24:36.700
We're not seeing lifespan get shorter. We're not seeing an uptick in cancer or something that was
02:24:41.180
unanticipated. So yeah, if you're neutral to positive on lifespan with these healthspan benefits
02:24:46.700
in terms of ejection fraction, periodontal disease, things like that, that would probably be
02:24:51.420
sufficient enough reason. Right. So now the ADAM study. Yeah. So this is a super interesting study
02:24:56.780
in a non-human primate called a marmoset. Marmosets are an interesting non-human primate model because
02:25:02.760
they aren't as long lived as rhesus monkeys. So rhesus monkeys, I think, in captivity will typically
02:25:07.480
live 30 to 40 years. Marmosets, I think this is a little bit of a moving target as people are
02:25:12.760
starting to use marmosets more in captivity. They're learning more about what the actual
02:25:17.040
life expectancy is, but it seems to be towards the, I think, low to mid-teens. So significantly
02:25:23.540
shorter lifespan in captivity. That makes them an interesting model as a non-human primate
02:25:29.980
to study aging. And so ADAM, for several years now, has had an ongoing marmoset colony in San
02:25:38.360
Antonio, some of whom have been getting rapamycin. And I don't think they've published the data from
02:25:43.700
that study, at least not the lifespan data. So I don't think it's complete, but they've already
02:25:48.860
published some preliminary data for bioavailability, blood levels, some interesting data, suggestive.
02:25:55.640
And Adam has talked in meetings about the apparent survival benefits so far, again, incomplete study,
02:26:03.640
where it looks like rapamycin may be having positive survival effects in marmosets. So again,
02:26:10.140
I think if that pans out and we actually see a statistically significant improvement in lifespan
02:26:15.540
from marmosets, that's really important because now it's gotten to the point of a primate,
02:26:20.280
which we don't have data for yet, obviously closer to humans. I think we also, though, have to
02:26:24.980
recognize there's still a pretty big limitation from that study in the sense that it was done
02:26:29.320
in captivity. So there are some questions about rapamycin, particularly, again, we've already
02:26:35.740
talked about, and I don't think any of us believe that rapamycin alone at lower doses is a potent
02:26:40.740
immunosuppressant. But when you're out in the real world and exposed to all sorts of environmental
02:26:46.300
challenges, it may be the case that the effects of rapamycin are going to be somewhat different
02:26:51.680
than what we see in the laboratory. So it's a huge step up, I would say, in the sense of being
02:26:57.180
in non-human primates, but it's still got that caveat that it's a laboratory-based study.
02:27:02.680
To me, one of the big issues with the mouse studies in rapamycin is that these are sedentary mice
02:27:08.040
who are getting fat, who are frankly depressed. And so what I always say about your study is the
02:27:12.800
critical aspect is that these are free-living animals who presumably are relatively happy. And so
02:27:17.340
the marmoset study sounds exciting, but it does have that big caveat of potentially more sedentary,
02:27:22.880
sad animals on occasion. I actually, frankly, hadn't even thought about the potential
02:27:26.900
infectious disease implications of it. But living in the human environment, to me,
02:27:32.580
is the key aspect that I'm looking forward to your study about.
02:27:35.900
I think it's hard to know how important those pieces are, but you're absolutely right.
02:27:39.640
You could imagine they're going to be hugely important. And so, yeah, it's just a difference.
02:27:44.040
What was the dosing in your study, Matt? Is it 0.1 milligrams per kilogram weekly?
02:27:49.600
No. So we've done two. The first one was, we tested two doses, 0.1 milligrams per kilogram,
02:27:55.480
three times a week, and 0.05. So half that dose, three times a week. And then we went to
02:28:00.960
0.15 milligrams once a week. And that's what we're using now. And we could talk about why we
02:28:08.860
made that decision. This is a challenge. Anytime you're trying to design a clinical trial,
02:28:12.860
there's sort of infinite number of variations on dosing and how you deliver and all of that.
02:28:18.120
We decided to go with that dosing protocol, 0.15 milligrams per kilogram once a week for the large
02:28:24.600
clinical trial based on the outcomes from the two shorter trials in terms of total dose,
02:28:30.960
so cumulative dose. And we haven't really talked yet about what sort of become popular in the,
02:28:36.820
for lack of a better word, biohacking community, which is this once weekly dosing.
02:28:40.460
But based on Joan's study, the observation that once weekly dosing with everolimus
02:28:45.460
seemed to give similar efficacy with maybe lower potential side effect risk. And from a pragmatic
02:28:52.180
perspective, because the owners are giving the drug to their dog, we thought that it would be
02:28:58.020
more likely that owners would be able to consistently remember to give the drug to their dogs
02:29:04.160
once a week as opposed to three times a week. And that's speculation, but all of those things
02:29:08.320
kind of weighed in to that decision. And I'm hoping it doesn't come back to bite us on the ass,
02:29:12.760
but that's the challenge with designing trials.
02:29:15.540
And that's where I'm going with it is if you sort of try to triangulate between
02:29:18.720
the everolimus study, your studies, Adam's studies, and the ITPs, you sort of coalesce around 0.1
02:29:32.200
milligrams per kilogram weekly for a human, which is kind of putting people, put someone my size,
02:29:41.240
maybe a bit more, but it's probably like in the 8 to 12 milligram range for someone our size.
02:29:46.940
I'm not sure actually about that. So this is actually, I was going to say one of my biggest
02:29:51.140
concerns with our dog study is that our dose is too low. We have to go low because you're trying
02:29:56.880
to weigh risk reward. And in people's pets, the tolerance for risk is extremely low. But I am
02:30:03.480
concerned that because we need to be so risk averse that we're having to dose lower than what might be
02:30:10.060
the optimal dose. And my real concern is we're dosing below what would be the dose you would need
02:30:14.940
to see any statistically significant effects. So that's my concern. I don't have any data to point
02:30:19.980
to, to suggest that. And actually I have some data to suggest that even at the doses we're using
02:30:24.640
in the two shorter trials, we did see evidence for beneficial effects, but that's kind of the thing
02:30:29.900
that keeps me awake at night. When I think about the design of this trial, the mouse studies, again,
02:30:34.180
I need to go back and really do this conversion, but my recollection of the mouse dosing was that it
02:30:38.840
actually works out something close to 0.1 mg per kg per day in people, not per week.
02:30:44.880
That's absolutely correct. It was 1.4 mg per kg per day is what the mice were actually given in the
02:30:52.580
ITP, which works out to when you convert that to human dosing, which is there's a conversion factor.
02:30:58.600
It works out to 0.1 mg per kg per day is what they were getting if they were humans. So you're right.
02:31:05.740
They were getting much more rapamycin. And yes, that speaks exactly to the concern we have,
02:31:13.420
which is how do you know if you're getting enough? And the only reason I think we may still settle on
02:31:20.200
this weekly dose is we at least saw the positive immune modulation with 5 mg a week of Everolimus.
02:31:27.980
I mean, that's even less. All I'll say is that to me, one of the fundamental differences from what I
02:31:33.780
hear and I have no particular expertise is that the mouse study is really our chronic dosing.
02:31:38.840
And really the best evidence is the manic study for an intermittent dosing having a clinical output
02:31:45.660
that's beneficial. I know there are mouse studies that have done that as well, but in some of these
02:31:49.440
larger animals. Yeah. So I think that's right. I'd say there's two pieces, right? One is the dosing
02:31:54.800
when you think about like daily versus weekly versus every other week, that sort of intermittent
02:31:59.960
dosing. But then there's the question of interval of dosing. How long do you need to dose? And so
02:32:04.800
those are two different variables that I think are both poorly unexplored, even in the mouse studies,
02:32:10.440
to really tease out where you see different benefits or where you get the biggest benefits.
02:32:16.540
What dose ranges are you seeing in the wild? So when you did that survey, what were you seeing?
02:32:21.920
What percentage of the RAPA users were on weekly doses versus daily doses versus tri-weekly?
02:32:27.600
And what was the range of the actual dose? I think in a general sense, it was kind of all
02:32:32.600
over the place, but by far the majority were once a week. And among those, the vast majority were six
02:32:39.840
milligrams once a week. And I think there's some historical reasons for that. That's become popular
02:32:44.640
in the online sort of community where people talk to each other. But where do you think that number
02:32:49.640
comes from? So I think it's partly from Joan's study. So it's close to Joan's study, which was five
02:32:54.800
milligrams once a week of Everolimus. I think it's also because many of the people,
02:33:00.300
the first people to start taking rapamycin off-label were patients of Alan Green out of New
02:33:05.420
York. And I think that was sort of his standard dose that he put most people on. So I could be
02:33:11.540
wrong about that, but that's my impression is that's kind of how that became popularized,
02:33:16.180
for lack of a better word, within the community. But having said that, there's a lot of variation
02:33:20.800
around that, both in terms of doses that people are taking once a week. And then there's a fraction
02:33:26.040
of people who are taking rapamycin daily, usually one milligram, two milligrams, sometimes for
02:33:33.520
purposes other than purely for aging. So people who have existing autoimmune disorders, sometimes people
02:33:40.980
are taking rapamycin for that. But in general, I would say among the off-label rapamycin users,
02:33:46.160
the majority are once a week. And of those, the majority are six milligrams. It's kind of a
02:33:52.280
bimodal distribution. There's a group of people around three milligrams as well, but lots of
02:33:56.620
variation around that. What were some of the higher doses you saw for the once weekly folks?
02:34:01.520
So I think our highest was up close to 20 milligrams once a week. Again, though, it was a little bit
02:34:08.260
difficult to tell how long people had been taking rapamycin at those doses. Again, some people had been
02:34:15.000
taking it, I think the person who's been taking it the longest was many years, I don't know about many,
02:34:19.840
five or six years. So, but the majority were six months. These people who reported taking 20
02:34:25.280
milligrams once a week could be that they just started doing that. And a lot of people, I mean,
02:34:30.620
as you know, right, a lot of these are N of one experiments with people who are changing their
02:34:34.380
regimens as they go. So there are some people who are taking six milligrams once a week, but they're
02:34:39.600
trying to build it up to some higher dose to see where they start to get side effects.
02:34:43.420
There are a bunch of people who reported taking grapefruit juice with their rapamycin because
02:34:48.060
grapefruit juice will inhibit cytochrome P450s and enhance bioavailability of rapamycin. So,
02:34:53.660
and I think the reason why I say that is both to illustrate the sort of nature of complexity of
02:34:58.660
this population, but also because we know there's going to be genetic variation in uptake of rapamycin and
02:35:06.400
how quickly the drug is metabolized. And so the dose that somebody is taking may or may not really
02:35:11.980
reflect the total bioavailability or the kinetics trough level, peak level, things like that.
02:35:17.180
One thing I would caveat folks who are listening to this, who are themselves taking rapamycin is,
02:35:22.140
first of all, it's not a cheap drug. So I think the most competitive pricing you'll find if you're
02:35:26.800
using good RX is works out to about $5 a milligram. Does that sound about right?
02:35:33.420
And so what you're seeing a lot of are these compounding pharmacies that are saying,
02:35:36.740
well, heck, I can just make this for you. I'll make it for you instead of giving you rapimmune,
02:35:41.820
which is even more expensive. That's the brand name. Rapimmune is the drug made by Pfizer or
02:35:46.620
just generic sirolimus. But I would caution people against using any compounded formulations here.
02:35:53.320
Yes, they'll make it a lot cheaper, but you have virtually no guarantee of the purity or the
02:35:58.160
concentration. We're already taking a huge leap of faith with this. We'll have a podcast that covers
02:36:04.000
the ins and outs of compounding pharmacies. I'm not here to suggest they're all bad, but
02:36:08.660
you absolutely want to be able to make sure you have FDA certificates for what you're using and
02:36:12.320
just be careful with the quality control. I would just add on to that as well,
02:36:17.140
particularly with rapamycin and compounded rapamycin. There is some data out there
02:36:21.160
on compounded rapamycin having essentially no bioavailability if it's not in an enteric capsule.
02:36:27.020
So this actually goes back to the reason why the ITP took 20 months to start their experiment as
02:36:32.600
rapamycin is unstable at gastric pH. And so if compounded rapamycin is not in an enteric
02:36:39.720
coated capsule, you're essentially going to get zero bioavailability.
02:36:44.120
This is one where I think you splurge and get either sirolimus or rapimmune.
02:36:48.480
Last thing I want to kind of talk about on the potential interesting front, it's the real tragedy
02:36:54.580
of not, I always say this in Matt, you and I and David, we've all talked about this. If I was a
02:36:58.760
billionaire, what would I do? I'd literally just set up a research institute that would fund this type
02:37:04.440
of work with no profit motive because nobody would care to fund this if you were profit-driven. But
02:37:09.680
the fact that no one's really looked at the impact of rapamycin on ovarian aging is really frustrating.
02:37:18.560
And by the way, you could also look at the impact of rapamycin on dermatogenesis,
02:37:22.240
but just we're reproducing at a later and later age in life. And fertility is such an important
02:37:28.260
part of that, especially as we experience population decline. So anything that we could
02:37:33.520
do to better understand how to preserve the youth of sperm and egg would be really fascinating.
02:37:40.800
I think there is someone looking at this in Brazil and someone looking at this here in the U.S.
02:37:46.260
I haven't heard enough from it. Maybe you guys know about this more. Do we know anything yet about
02:37:52.200
this? Yeah. So again, we can go back to the mice and it's pretty clear in mice, female mice, that you
02:37:58.260
can delay or probably even reverse ovarian atrophy up to some point in life with transient rapamycin
02:38:06.080
treatment, actually restore reproductive capacity to sterile female mice through this sort of treatment.
02:38:11.820
It's interesting though, in male mice, it's the opposite. So you actually seem to impair
02:38:16.900
spermatogenesis and potentially induce sterility. It's worth just noting that there may be differences
02:38:23.140
in the male and female reproductive systems there. Why do you think that is?
02:38:27.400
I think it goes back to what I mentioned before. If you ask what cells are most impacted by rapamycin
02:38:32.940
in vivo, what is their defining set of characteristics? It's always the most rapidly proliferating.
02:38:39.860
And that, I think, is what defines spermatogenesis versus oogenesis, which by definition is amongst
02:38:46.140
the slowest processes we have. You need a certain rate of growth, anabolism, when you're proliferating
02:38:52.060
quickly that you just don't need when you're proliferating slowly, and therefore you impact
02:38:56.620
those cells. So I think you're right, Matt. From the studies I've looked at, clearly spermatogenesis,
02:39:01.820
male fertility is negatively impacted. There's no doubt about that.
02:39:04.960
At least well on rapamycin. I think there's at least one paper that showed that once the
02:39:10.060
mice come off of rapamycin, there may actually be a preservation of sperm quality in male mice. So
02:39:15.020
again, this gets back to dose and duration and transient versus continuous. I think you're
02:39:20.360
probably right about that, the mechanism potentially just boiling down to cell cycle.
02:39:24.680
And the ovarian one, I always wonder, and this is in general what I always wonder about rapamycin and
02:39:29.300
sort of its potentially anti-aging properties, is how much is simply a delay because you're
02:39:34.800
slowing the cell cycle and the progression of cells versus a true rejuvenation. And so
02:39:39.160
you mentioned a true rejuvenation, and that is very impressive. That's what you're seeing.
02:39:43.300
So this is not my data, and I don't know that the papers have been published yet,
02:39:46.880
but they've been presented at multiple meetings. There's one that just struck me as so profound,
02:39:51.200
where you can actually see structural rejuvenation of the ovaries from an
02:39:54.980
atrophied state to at least at a morphological level appears like true rejuvenation,
02:40:00.460
as well as a restoration of fertility. Of the actual ovary or of the oocyte?
02:40:05.300
The ovary. Wow. Which is hard to understand, right? Because presumably when you're fully
02:40:09.240
atrophied, you have no oocytes left to rejuvenate. Well, I don't know about fully. This is where I
02:40:14.020
think it probably depends on how far down the path you've gotten. I don't know that that's been even
02:40:18.460
carefully done. Has anybody looked at rapamycin administration and anti-malarian hormone level
02:40:24.700
for example? Once, let's say, a woman is already in her AMH decline, but hasn't fully bottomed out
02:40:31.460
to zero, could you rescue some of that? I mean, because if you look at the physiology of that,
02:40:36.480
it is a monotonically decreasing function, and it is very steep. And if you could simply stop it
02:40:43.180
from declining, that would be remarkable, let alone turn it in the other direction. Again,
02:40:48.040
here's an example of you could study this for hundreds of thousands of dollars. These are not
02:40:53.180
large sums of money. It would have to be paid for by somebody who's just genuinely obsessed with
02:40:59.180
knowing the answer and not realizing they can't make a buck off this.
02:41:02.880
Right. We talked about the human studies that are going on now. So the one I'm most familiar with is
02:41:07.480
out of Columbia. So Yuxin Tzu, who directs the Reproductive Aging Center at Columbia, and Zev
02:41:13.740
Williams are leading this clinical trial. That's one of the biomarkers that they're looking at. I don't
02:41:18.280
think they have any data yet. And this was a grant that was funded by the Impetus Grants Foundation.
02:41:23.500
So these are smaller grants. But like you said, you can actually start to answer some of these
02:41:28.600
questions. And it doesn't require tens or hundreds of millions of dollars to start to gather some data.
02:41:33.780
So they have a clinical trial that is ongoing. I don't know. I've actually got a call with them
02:41:39.220
next week. So I don't know how far along they are, except I do know that there are some patients in
02:41:43.480
the trial now looking at women with premature ovarian failure. And it's a double-blind,
02:41:50.960
placebo-controlled, randomized clinical trial with rapamycin. So I think that will start to
02:41:56.460
get more data around safety in a younger, healthier population and hopefully start to get
02:42:02.260
some data around potential efficacy for ovarian function in people. I want to just mention,
02:42:08.700
though, I mean, I'm extremely pleased that the Impetus Grants Foundation funded that trial.
02:42:12.420
They're also funding a periodontal disease trial out of the University of Washington.
02:42:16.080
I'm grateful to them for doing that from like a scientific perspective. But I'm also extremely
02:42:21.220
frustrated that the funding for these kinds of trials is so small. And these trials are,
02:42:27.380
let's be honest, they're underpowered for what we would really want to do. If you really want to
02:42:32.100
answer the question, you're not going to get there with trials of 40 people. That's not enough.
02:42:37.720
The researchers are doing the best they can in the system that we're working within. But what happens
02:42:43.860
is you end up with these small clinical trials that give a hint of efficacy and show no problems in
02:42:49.620
terms of safety. But then it takes another two years, another three years, another four years to
02:42:54.680
get a grant to take it to the next stage. And that's why it ends up taking two decades with something
02:42:59.440
like rapamycin to actually get to an answer. Whereas if this was a rapalog, an mTORC1 specific
02:43:06.400
rapalog, you could go out, start a company and raise money to do a more accelerated path. I don't mean
02:43:13.420
to dismiss that approach. But we have a perfectly good drug here with lots of human safety data
02:43:18.460
that probably works. And it's frustrating to say the least that things have gone so slow.
02:43:24.100
It really is because people don't appreciate what it takes to go from IND to phase three approval.
02:43:31.620
And the fact that that's already been done for this molecule, and basically all you're really
02:43:37.400
doing is a series of new phase two and phase three trials on an approved drug is such an enormous
02:43:44.880
tailwind. I share your concern. David, one kind of last thought from you. You're quite close to the
02:43:52.040
landscape of this. You've personally been involved in the development of a number of rapalogs. What is
02:44:00.540
your take today of the landscape in this arena? It's so funny how the winds change. I would say
02:44:07.480
10, 12 years ago, if you went and said, I want to target mTOR, the universal response was,
02:44:13.140
we have rapamycin, it's off patent, it's cheap. No, thank you. Let's move on. I think now there's
02:44:19.220
almost been a complete reversal as Matt kind of alluded to, right? You can come up with small
02:44:24.220
differences in rapamycin, which you can still sort of patent protect and apply them to much more niche
02:44:30.080
applications and the people are potentially willing to fund them. These are not the mega biotechs that
02:44:35.160
are being started, but certainly enough to get things going. And so I think there's a general
02:44:39.780
consensus that mTOR matters as a target. What is frustrating to me, if Matt mentioned his frustrations,
02:44:45.600
sure, I think rapamycin and its derivatives are great, and we should do exactly what Matt was
02:44:50.200
saying and somehow incentivize that. I personally think, though, there's actually a whole bunch of
02:44:54.200
other targets in that pathway that may be more beneficial. For example, one thing we didn't get
02:44:58.940
to here, and Matt alluded to this, it's very clear that the response to nutrient deprivation is not just
02:45:03.960
mTOR at all. In fact, the nutrient sensors we found clearly talk to a whole bunch of other
02:45:09.640
processes. And so if you want to get closer to that nutrient deprivation state, one has to go to
02:45:15.400
those. And so right now, the way I read it is people are willing to invest modestly in molecules
02:45:22.120
that are rapamycin derivatives. They still, though, have the mindset mTOR is drugged. And therefore,
02:45:27.780
if you want to go to other components of the pathway, which I think we don't have time to discuss
02:45:32.120
them, would be more interesting. There's really not an appetite for that.
02:45:36.320
Gentlemen, I have a list of pages in front of me of topics that I wanted to discuss with you that
02:45:44.040
extend far beyond rapamycin and mTOR. We've very, very briefly touched on a couple of them. We've
02:45:49.700
talked a little bit about epigenetics. We've talked a little bit tangentially about some of the other
02:45:54.380
hallmarks of aging. We've had hints of other questions that are remarkable, questions that seem
02:46:00.180
so basic and fundamental, and yet it's amazing we don't know the answer to them. Questions such as,
02:46:04.260
why do different organisms age at different rates? Why do different organisms of similar size have
02:46:09.900
different lifespans? These are all some of the most interesting questions in biology and questions
02:46:13.940
that we collectively as friends discuss all the time privately. But I think it would be really
02:46:19.740
enjoyable to have one of these discussions publicly in this way. So I think the only thing to say here is
02:46:24.960
we should just probably sit down collectively and do this again, much sooner than when we plan to go
02:46:30.680
back to Rapa Nui, which guys, I have that on the calendar for 2026. Let's find time between now and
02:46:37.300
then to sit down and do a part two of this discussion, which I hope was half as enjoyable for you guys as
02:46:42.300
it was for me. It was great, Peter. Thank you so much. It's always so much fun to talk to you and Matt
02:46:46.780
to hear all your thoughts. Thank you. Yeah. Anytime, guys. This has been a blast. I like the three-way
02:46:52.440
podcast here, Peter. It works. Yeah. Or we could do it in person the next time, guys. It's even more fun in
02:46:57.500
person. Definitely. Yeah. I remember, Peter, when we go back to Easter Island, we got to bring the
02:47:01.980
plaque. I know. We are bringing the plaque. We went to the place where the soil sample was collected.
02:47:07.260
There's supposed to be a plaque and the plaque was stolen. So we're going to do that. Yeah, we will
02:47:12.340
indeed. All right, guys. Thank you so much. Thank you. Thank you. Thank you for listening to this
02:47:17.740
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02:47:22.720
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