The Peter Attia Drive - September 25, 2023


#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

Word Count

31,065

Sentence Count

1,703

Misogynist Sentences

2

Hate Speech Sentences

8


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

00:00:00.000 Hey, everyone. Welcome to The Drive Podcast. I'm your host, Peter Atiyah. This podcast,
00:00:16.580 my website, and my weekly newsletter all focus on the goal of translating the science of longevity
00:00:21.580 into something accessible for everyone. Our goal is to provide the best content in health and
00:00:26.780 wellness, and we've established a great team of analysts to make this happen. It is extremely
00:00:31.720 important to me to provide all of this content without relying on paid ads. To do this, our work
00:00:37.000 is made entirely possible by our members, and in return, we offer exclusive member-only content
00:00:42.720 and benefits above and beyond what is available for free. If you want to take your knowledge of
00:00:47.980 this space to the next level, it's our goal to ensure members get back much more than the price
00:00:53.240 of the subscription. If you want to learn more about the benefits of our premium membership,
00:00:58.080 head over to peteratiyahmd.com forward slash subscribe. My guests this week are David Sabatini
00:01:07.140 and Matt Caberlin. Matt has been a former guest on three occasions, episode 222 and 175, and all the
00:01:15.320 way back to episode 10, while David was on the podcast way back in episode number nine. Not only
00:01:22.780 are they two of the original guests on the podcast from our 12-part pilot experiment in the summer of
00:01:29.660 2018, they are also two of the most knowledgeable people on this planet on the topic of conversation
00:01:37.240 today, rapamycin and mTOR. In my conversation with Matt and David, we cover the discovery of
00:01:44.140 rapamycin. We look at how mTOR, which sits at the epicenter of our existence, works and does its job.
00:01:50.160 We talk about the pathways of rapamycin that are believed to drive its impact and improvement on
00:01:55.140 lifespan. We discuss the initial studies that showed rapamycin may be gyroprotective, and also
00:02:01.180 what studies have come out since or are currently in progress, which provide more information and
00:02:05.800 clarity around this very important question. Finally, we discuss what is known and unknown about
00:02:11.560 the potential frequency and dosing of rapamycin in humans. I'm very excited to release this podcast,
00:02:18.700 because I don't think there is a question I get asked more about from my patients than this topic.
00:02:25.940 Understandably, because my patients know that I take rapamycin and have been doing so for about five
00:02:31.520 years, it's understandable that they want to understand if it's something they should be doing
00:02:35.880 too. And I think you'll see from this discussion why I have reservations about just blindly putting
00:02:41.380 people on rapamycin. In other words, why is our practice not a rapamycin mill? I hope that this
00:02:47.620 podcast is helpful, not just for my patients, but everybody listening, including everyone who is
00:02:52.960 interested, of course, in this question. So without further delay, please enjoy my conversation with
00:02:58.780 David Sabatini and Matt Cabron. Well, guys, we're going to try something a little different today,
00:03:09.620 which is we're going to try to have a three-way discussion, which is something we would easily
00:03:16.040 be doing if we were sitting over a meal, but always makes for a slightly more challenging podcast.
00:03:21.580 That said, given our familiarity with each other and your familiarity with this topic, I am
00:03:26.840 a hundred percent confident this is going to be an amazing episode. It's also an episode that is
00:03:31.080 long overdue. So you guys are both in the camp of, I believe, first dozen or so podcasts that were
00:03:40.300 released on the drive a little over five years ago. Matt, you and I have spoken a number of times
00:03:45.320 since. David, you and I, at least on the podcast, have not, obviously, in person all the time. So with
00:03:50.740 all that said, many people are going to be new to this topic. They will have heard a lot about it.
00:03:56.000 They may have even read a chapter about it in my book, which you guys were both very gracious to
00:04:00.320 help me fact check and edit. But here we are. We're going to pretend that someone coming into
00:04:05.160 this discussion doesn't really know anything about rapamycin, doesn't really know what this
00:04:08.560 mTOR thing is. I hope that by the end of this discussion, we will have provided people with
00:04:14.020 arguably the most comprehensive, quasi-concise explanation of all you need to know about said topics.
00:04:23.600 With that said, I would like to ask each of you to do something I don't often ask my guests to do,
00:04:30.960 which is toot your own horns a little bit about what it is that allows me to say you are each
00:04:38.180 among the two most knowledgeable people on this topic. Let's start with you, David. You've worked on
00:04:45.180 this molecule, rapamycin, your entire scientific career going back to your PhD. And here we are
00:04:52.940 30 plus years later, you're still the leading authority on it. Can you tell us a little bit
00:04:58.260 about that? Sure, Peter. Thank you for having us. Nice to see you, Matt. So no, indeed, I've worked on
00:05:04.060 rapamycin my whole life. When I was a student with Saul Snyder or Johns Hopkins, I became fascinated with
00:05:08.980 this molecule. And frankly, I needed a research topic. And so I tried to figure out how it works.
00:05:13.820 And that led to the purification of protein that we now call mTOR. Michael Hall had identified a
00:05:19.780 yeast version of this that Matt was one of the early workers on this called TOR1. And since that time,
00:05:25.760 we've done a lot of biochemistry, try to figure out what this protein does. And at the end of the day,
00:05:29.920 what we conclude in a kind of big picture point of view is that this is the protein that links
00:05:34.680 the availability of nutrients in our environment to whether we're in a catabolic or an anabolic
00:05:40.240 state. Anabolism growth, catabolism breakdown of material. And I think that accounts for why mTOR
00:05:46.920 has so many different roles. Because if you think about our sort of evolutionary history,
00:05:51.100 there's almost nothing in our physiology that shouldn't be controlled by the availability of
00:05:54.820 nutrients. It's such a central thing in our lives. We tend to forget that now because of course,
00:05:59.640 we're in a overeating stage. And since that time, what we've done is figured out a lot of the pieces
00:06:04.520 of this pathway, including what we call two complexes, protein complexes, mTORC1 and mTORC2.
00:06:11.060 And really the work that I'm the most satisfied with is how it senses nutrients and the nutrient
00:06:15.720 sensors themselves, which are the actual proteins that bind the small molecules that tell mTORC1 in
00:06:21.900 particular that it detects nutrients. And so I'm excited to be here and to delve into some of the
00:06:27.420 implications of this work. Awesome. Thanks, David. Matt, people who are listeners of this podcast are
00:06:33.980 going to be maybe a bit more familiar with you because in addition to that very first podcast
00:06:38.400 we did circa 2018, you've been back a number of times and we've talked about mTOR and rapamycin,
00:06:45.640 but we've also talked about protein, nutrition, and things like that. But maybe for folks who are
00:06:49.700 hearing you for the first time today, can you give a similar bio of what it is that allows me to
00:06:56.680 also refer to you as one of the world's absolute leading authorities on this topic?
00:07:01.280 Sure. First of all, thank you for having me back. I'm glad you aren't sick of me yet. It's fantastic
00:07:06.860 to be here with David. And I wish I could say that I was smart like David was and recognized
00:07:12.820 immediately how important mTOR would be and rapamycin. But I actually started working on mTOR
00:07:18.980 in yeast kind of by accident. So we were really interested in understanding what are the genetics
00:07:25.060 that control longevity. And so we did an unbiased search for new genes that would affect lifespan
00:07:31.600 and happened to find mTOR. And when we made that discovery, I immediately went and looked up
00:07:39.080 everything I could learn about mTOR and found out there's this drug, rapamycin, that's an inhibitor
00:07:44.100 of mTOR. And then we found that we could also increase lifespan with rapamycin. At this point,
00:07:49.400 we were working in yeast. But then it became clear to me because of the work of others that this pathway
00:07:56.480 and this drug appeared to affect the biological aging process, not only in yeast, but also across
00:08:03.200 the animal kingdom. And we now know even in mammals like mice and potentially in larger mammals like dogs
00:08:10.260 and people. So I think with that knowledge, I got very interested in trying to understand what the
00:08:16.340 mechanisms are for how rapamycin was affecting the biological aging process. We've studied this in
00:08:23.220 yeast and worms and fruit flies and mice, a little bit in pet dogs, which we may talk about. And I think
00:08:29.260 through all of that, the one thing that has kept me excited about rapamycin as a potential longevity
00:08:35.500 therapeutic is that it always works. And I would say without question, it is the most robust and
00:08:41.680 reproducible drug, at least from preclinical studies that we know about today for impacting
00:08:48.120 not only longevity, but to the extent that we can measure various metrics of health span
00:08:53.300 in complex animals, rapamycin also seems to positively impact pretty much every aspect of
00:08:59.040 health span that we measure. So I've continued to study it for that reason. And I think probably what
00:09:04.020 I'm maybe best known for these days is pushing forward a veterinary clinical trial of rapamycin to really
00:09:10.180 start to answer the question of all the things we've learned about rapamycin in the context of
00:09:15.000 aging and longevity in the laboratory, how much of that will translate into the real world? And so we are
00:09:19.900 actually carrying out a veterinary clinical trial of rapamycin in pet dogs right now. We've got some
00:09:26.280 preliminary data, but it's too early to be able to say, you know, with any level of confidence that
00:09:30.380 rapamycin is going to positively impact the aging process in dogs. But I think we've already learned a lot
00:09:35.400 about safety and maybe some hints about efficacy. So that's pretty exciting. And that's something that I
00:09:39.660 quite passionate about continuing to push forward and see where we end up.
00:09:44.240 And I guess I should, before I make my next comment, disclose that I and a number of my patients are
00:09:50.160 funders of a study that we will undoubtedly talk about. So we should just, I guess, declare that
00:09:55.260 as a conflict if people want to consider that a conflict. But regardless, I think what's really
00:10:00.000 great about having you guys together, and tell me if you agree with this assessment, is on the
00:10:04.780 continuum of understanding rapamycin and mTOR, David, you're closer to what we would call the bench
00:10:12.520 side of things. And in many ways, Matt, I consider you kind of closer to the bedside. So people have
00:10:18.040 heard this term bench to bedside, i.e. translational research. And obviously the bedside in this case is
00:10:24.420 not just the bedside of humans where we aren't quite yet, but really the bedside of more complex mammals.
00:10:30.060 So would you guys kind of agree with that assessment that your skill sets and your knowledge
00:10:34.220 base and your research are very complementary through that continuum of bed-to-bedside?
00:10:39.400 I would. I would add, though, that Matt takes the work in a very serious scientific way, right? And so
00:10:45.220 I think in a field where it's very easy to get caught up in boosterism and claims that you see online
00:10:50.840 all the time that are extreme, I think Matt has been very careful. And part of what's contributed
00:10:56.260 to the interest in rapamycin, frankly, is that Matt has been careful about this. And so he's taken a
00:11:02.920 very scientific approach. And as I've told many people who know me well, I pretty much put Matt
00:11:07.940 in one of the most respected categories of aging researchers for that reason.
00:11:12.540 I agree with that completely.
00:11:14.060 You guys are embarrassing me. Come on now.
00:11:17.080 Peter, let me just give a little bit of a twist on what you said. I mean, I agree with what you said
00:11:21.120 conceptually. I would also say, I think even though much of my research on mTOR and rapamycin has been
00:11:26.740 what people would typically consider preclinical or basic research, it is different. I agree. I think
00:11:32.460 the approach that David has taken throughout his career is quite complementary in many ways to the
00:11:36.520 approach that I've taken in that David has really, as you already said, been the pioneer and the leader
00:11:42.080 in really understanding detailed mechanistic aspects of the whole mTOR signaling network.
00:11:50.380 And I don't know how much we're going to get into that, but I think it's useful for people to
00:11:53.840 appreciate that this is an extremely complex network of biological interactions. And there's
00:12:00.120 no question that David and his lab and people who have come from his lab have really played the
00:12:06.020 leading role at fleshing out from a very detailed biochemical and mechanistic perspective, how that
00:12:11.680 network is working. And that has, I think, in many ways laid the foundation for people like me and many
00:12:17.880 others who have then taken that knowledge and tried to start to move it into maybe more applied
00:12:23.340 contexts and clinical applications.
00:12:26.260 That's a beautiful way to describe it. And I also want to echo something that David just said. And again,
00:12:31.920 not to embarrass you, Matt, but I do think that the field owes a lot of its credibility to the way you have
00:12:39.180 approached it with scientific rigor being the highest priority, as opposed to the commercialization
00:12:45.220 not being front and center. And I do think that there are a lot of other molecules that maybe we
00:12:51.240 don't have to get into today, where there is some interesting science behind it, but it seems to have
00:12:56.640 almost been corrupted by a commercialization route. And the corruption of that has meant, A, we may never
00:13:05.220 know if these things work or don't work, but more than anything else, they're very difficult to take
00:13:10.440 seriously. And I think everybody should be very grateful for the way the field has gone.
00:13:14.340 So before we dive into it, and I know this is a story that's been told before on this podcast,
00:13:19.180 and I probably even write about it in the chapter of my book, I do think that the discovery of
00:13:24.340 rapamycin is the place to begin this, because there's a very unique phenomenon here, which is
00:13:31.480 the drug was discovered before the target, and the target is named after the drug in response to that.
00:13:38.320 So David, you and I got to visit this very special place where the bacteria that ultimately
00:13:44.000 produced this drug was discovered. We certainly have plans to go back. It's on our list of things
00:13:48.240 to do in the next few years, and we shall. Do you want to give folks the story of how this
00:13:52.720 molecule rapamycin came to be?
00:13:55.100 Sure, sure. And I hope we'll go back soon, and I hope Matt will come as well.
00:13:58.220 He certainly will.
00:13:59.540 Anyone who cares about rapamycin.
00:14:00.840 For the record, I did try to get this recording done on Rapa Nui, so I just want to put that out there.
00:14:06.340 We'll be doing another one there for sure.
00:14:07.940 As you know, Peter, there were, in our attempts by pharmaceutical companies to collect soil samples
00:14:13.220 and other biological containing samples throughout the world, and their Wyeth Ayers did come into
00:14:19.380 possession of a soil sample from Easter Island, otherwise known as Rapa Nui in the South Pacific,
00:14:24.940 at one point claimed to be the most remote island of the world. I think it's actually technically not,
00:14:29.300 but very far from anything. And in that soil sample, actually in Canada, people eventually
00:14:34.480 isolated bacteria from it. Bacteria called Streptomyces hygropagus. And from that bacteria,
00:14:41.100 rapamycin, was eventually isolated, and in deference to Rapa Nui was named rapamycin.
00:14:46.100 Now, ironically, it turns out when people have looked for rapamycin and other bacteria throughout
00:14:51.100 the world, and in fact, even the same bacteria, it actually has been found in many other places,
00:14:54.540 but it did come originally from rapamycin. And like was done at the time, these molecules,
00:14:59.960 these bacterial products, you really would call an antibiotic, it did come from bacteria,
00:15:05.380 were tested, you know, in many different assays. And I actually think, and Matt may correct me,
00:15:09.020 I think some of the earlier assays were actually immunological assays, even before some of the
00:15:13.820 antifungal assays. And that eventually led, many decades after, to pursuing it as an immunosuppressant,
00:15:20.040 but in the meantime, it was also found to have antifungal agent activity. And that's where some
00:15:24.540 of the genetics of rapamycin and some of the targets were first identified because of the ease of
00:15:29.040 genetics. So this is a story that began, I think the original soil samples may have even been in
00:15:34.000 the 60s. Yeah, I think it was 66 or 67 soil samples. And then Seren, I don't think really
00:15:40.000 got around to digging into it until 71 or 72. Exactly. And then he championed it. In fact,
00:15:46.780 one of my most valued possessions, when I started working on rapamycin, we didn't have much. And Saul
00:15:53.260 Snyder, my advisor, wrote Seren and asked for some. He sent us many grams, which I had laid
00:15:59.020 or calculated, had a street value of many hundreds of thousands of dollars if one could sell it like
00:16:03.860 that. And a really nice note wishing me luck. And the entire bibliography of rapamycin at that time,
00:16:08.860 which consisted of his papers and a couple of abstracts, is a little thin book at the time.
00:16:15.140 And he is the one who championed it. The clinical path took way too long. And I think that even
00:16:20.100 impacted some of its utility because the patents expired, I think, before you could really sort of
00:16:25.140 capture some of the value of it. So we're talking about something now that's in the 50-year range
00:16:30.780 plus. And I think a question that we could ask ourselves, and I think we will, is rapamycin as
00:16:36.040 good as it gets? There obviously are derivatives of rapamycin. But even in this pathway, which as Matt
00:16:40.840 says, exceedingly complicated, are there other targets that we should be pursuing that may actually
00:16:46.440 have equal or better impacts on the aging process? Can I just add one thought? Something David said
00:16:53.160 there we may again also touch on, which is the clinical path not only took too long, but I think
00:16:58.220 you can make an argument that the clinical path has actually maybe negatively impacted the development
00:17:04.760 of rapamycin and other mTOR inhibitors for other uses. Because it was developed clinically as an organ
00:17:11.640 transplant immunosuppressant, and that's how it was first approved, it was used in a dosing protocol
00:17:17.460 and a patient context where there are lots of side effects. And I think we are still learning
00:17:22.540 what the side effect profile actually looks like for rapamycin at lower doses in patients who are
00:17:29.220 not immunocompromised and haven't had an organ transplant. So I do wonder whether the history of
00:17:35.940 rapamycin and the rapidity at which it will be eventually tested for other endpoints in clinical trials
00:17:43.700 where it may have benefits has been negatively impacted and slowed down because of the reputation
00:17:50.400 that the drug got as a dangerous drug based on the way it was developed clinically. So I think that's
00:17:56.920 an important piece of the puzzle here to think about. Just to give some numbers to it, the first paper
00:18:03.180 that Soren Seagal put out there describing the chemical composition of rapamycin, if I'm not mistaken,
00:18:08.940 was about 1971, 1972, the FDA approval for rapamycin in humans was 1999. Just to give you a sense of what
00:18:19.720 you're both talking about here in terms of an enormous gap of time between when you sort of make
00:18:26.020 a chemical discovery, file an IND and work all your way through. Second point I'd make is as a former
00:18:32.180 surgical resident, I was in my surgical residency taking care of transplant patients when rapamycin
00:18:38.580 was in full use. Now, again, it's interesting, David, the whole reason you got involved in
00:18:43.820 rapamycin was because of FK506, which was a cousin of rapamycin that, if I recall, the whole reason
00:18:52.420 your lab was using rapa was as a control that didn't have sort of the calcineric properties of
00:18:58.620 FK506. But that's an interesting footnote. But we were giving rapamycin out constantly. And to your
00:19:03.520 point, Matt, it was a drug that was typically given two to three milligrams a day every single day
00:19:08.580 but with three other drugs. You were also getting prednisone, cell sept, MMF. You were getting
00:19:15.600 very, very toxic drugs because you needed to completely shut down the cellular immune system
00:19:21.900 of a patient who had just received a foreign organ. And I think that speaks to this point,
00:19:26.740 which is for the better part of a decade, 1999 to 2009, the only experience the scientific world has
00:19:34.480 with this is in that context. Yeah, you're going to see a lot of side effects, but how do you know
00:19:38.840 they're from rapamycin? And how do you know that they would be the same elsewhere? So what happened
00:19:43.460 in 2009 that kind of changed this? And David, I'm most interested, I think, in hearing this from you
00:19:51.420 because by this point, you've already established your own laboratory. You're working on rapamycin.
00:19:57.260 You're working on mTOR probably more so than anything else and trying to understand the nutrient
00:20:01.180 sensing pathways around it. But how aware were you of the ITP, the interventions testing program,
00:20:09.440 in the buildup to that first study in 2009? I was not very aware of it, I have to say. But I do have
00:20:14.440 to say that once we started making that connection of rapamycin to nutrients, which many groups did,
00:20:20.880 if you actually look at the history of it, and it was already appreciated for many, many decades before
00:20:25.560 that things like caloric restriction had an impact on lifespan. So the idea that rapamycin could have
00:20:31.820 an impact on lifespan was one we actually thought of. And we actually, this just tells you how science
00:20:36.820 works, we actually tried dosing C. elegans, worms, with rapamycin, naively not realizing that their
00:20:42.960 cuticle would not allow rapamycin the way that we were giving it to have an impact. We had found no
00:20:47.860 impact. And then there were genetics that came out in worms and Matt's work. And a lot of other people
00:20:52.760 really pioneered the aging space, not us at all. But I remember when that paper came out, I think it was
00:20:57.720 a nature paper that came out reporting rapamycin as one of the bigger hits in the ITP study. And I think
00:21:03.300 what happened there, I think Matt said this before, it connected his work in yeast and work in other
00:21:09.360 organisms with a mammal. Now, we just take that for granted, right? Because as Matt said, it does impact
00:21:14.480 all those different animals and single cell organisms. But the idea that we had a molecule that spanned from a
00:21:21.700 yeast to a mouse was dramatic. That was like a huge, huge impact. Again, we take it for granted now.
00:21:29.040 I'd like you to say more on that, both of you. I do think that the evolutionary gap from yeast
00:21:35.380 to flies, worms, mammals is a billion years. Are there any other molecules that have done what you
00:21:44.740 just said, David? I don't know if there are, but certainly dietary restriction, yes,
00:21:49.240 in one form or another. And that did link all those organisms. And as far as I know, it was all done
00:21:54.980 before rapamycin, before the discovery of TOR. So there was this universal intervention. I think
00:22:00.480 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
00:22:12.740 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
00:22:26.460 outside of caloric restriction, which by the way, doesn't universally extend life. There are models
00:22:32.300 and certainly times at which that can be administered when it is not a life extending strategy.
00:22:38.900 But yes, I mean, rapamycin in that sense stands alone. Unless, Matt, you can think of a
00:22:43.560 counterexample that I'm missing. I'm going to try to respectfully tell you guys that you're wrong.
00:22:49.300 So here's what I would say. Rapamycin for a small molecule is probably the only pharmacological
00:22:57.280 intervention that has been reproducibly shown to robustly increase lifespan and healthspan across
00:23:04.480 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
00:23:19.400 just add a little bit of additional context to what David was saying, which is that with rapamycin,
00:23:25.780 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
00:23:45.520 Daniel Promislow, this was probably 2007, where we asked the question, if we looked at all of the
00:23:50.680 genes at that time that were known to affect lifespan in yeast, and all of the genes that were known to
00:23:55.560 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
00:24:07.900 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
00:25:12.320 a lot of people have lost track of that.
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.
00:42:09.500 How uninteresting, right?
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.
00:43:16.960 Or mitochondria.
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.
00:47:14.280 What year was this, David?
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,
00:49:11.100 which we now do have that understanding.
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.
00:59:54.780 Was this Bobby that led this work?
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.
01:00:54.180 How long does leucine stay in that pocket?
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.
01:09:56.740 type of dosing.
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
01:15:01.020 physically large is it?
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
01:15:13.960 more in the 2 to 3 to 400 range. This is big.
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.
01:15:27.280 It's more about the solubility than size.
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,
01:32:14.020 is a very interesting footnote to the story.
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.
01:44:17.620 So in a sense, that's a nice positive control.
01:44:20.120 I was just about to say, it's my only biomarker that I know that I'm getting
01:44:23.620 high quality rapamycin.
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
01:44:38.040 at five milligrams weekly. It was like 15%.
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
01:45:35.600 it down and perhaps losing barrier function.
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.
01:47:08.320 Yeah. No, it's interesting. A little FK paste.
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.
01:47:23.780 Completely agree. We don't know that.
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
01:47:50.640 and unexplored.
01:47:52.100 Talk to me about any of the immune stuff that you saw, because you happened to run this survey
01:47:57.640 during COVID. What did you learn there?
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
01:49:07.320 doing to mTOR?
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
01:49:17.540 ketamine study argued that as well.
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?
01:51:17.040 I don't know. Outside my area of expertise.
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,
01:51:38.840 they were less impacted by 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
02:04:48.180 system and then release and see what happens.
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.
02:05:01.320 And a dirty one.
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
02:05:25.500 correctly.
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
02:14:28.660 processes.
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
02:18:57.360 mice live or how long C. elegans live.
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,
02:23:06.440 I'll take rapamycin.
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?
02:35:32.220 Yeah, that's 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 week's episode of The Drive. It's extremely important to me to provide all of this content without
02:47:22.720 relying on paid ads. To do this, our work is made entirely possible by our members. And in return,
02:47:28.500 we offer exclusive member-only content and benefits above and beyond what is available for free.
02:47:34.680 So if you want to take your knowledge of this space to the next level, it's our goal to ensure
02:47:38.560 members get back much more than the price of the subscription. Premium membership includes several
02:47:43.780 benefits. First, comprehensive podcast show notes that detail every topic, paper, person,
02:47:50.500 and thing that we discuss in each episode. And the word on the street is nobody's show notes rival
02:47:55.900 ours. Second, monthly ask me anything or AMA episodes. These episodes are comprised of detailed
02:48:03.060 responses to subscriber questions, typically focused on a single topic and are designed to offer a great
02:48:09.020 deal of clarity and detail on topics of special interest to our members. You'll also get access to
02:48:13.940 the show notes for these episodes. Of course, third delivery of our premium newsletter,
02:48:19.140 which is put together by our dedicated team of research analysts. This newsletter covers a wide
02:48:24.720 range of topics related to longevity and provides much more detail than our free weekly newsletter.
02:48:30.740 Fourth, access to our private podcast feed that provides you with access to every episode,
02:48:36.600 including AMA's sans the spiel you're listening to now and in your regular podcast feed. Fifth,
02:48:43.200 the qualies, an additional member only podcast we put together that serves as a highlight reel featuring
02:48:49.700 the best excerpts from previous episodes of the drive. This is a great way to catch up on previous
02:48:54.860 episodes without having to go back and listen to each one of them. And finally, other benefits that
02:48:59.880 are added along the way. If you want to learn more and access these member only benefits, you can head
02:49:05.280 over to peteratiamd.com forward slash subscribe. You can also find me on YouTube, Instagram, and Twitter
02:49:12.300 all with the handle peteratiamd. You can also leave us review on Apple podcasts or whatever podcast
02:49:19.320 player you use. This podcast is for general informational purposes only and does not constitute
02:49:24.940 the practice of medicine, nursing, or other professional healthcare services, including the giving
02:49:29.460 of medical advice. No doctor patient relationship is formed. The use of this information and the
02:49:35.780 materials linked to this podcast is at the user's own risk. The content on this podcast is not intended
02:49:41.820 to be a substitute for professional medical advice, diagnosis, or treatment. Users should not disregard
02:49:47.420 or delay in obtaining medical advice from any medical condition they have, and they should seek
02:49:52.000 the assistance of their healthcare professionals for any such conditions. Finally, I take all conflicts of
02:49:58.220 interest very seriously. For all of my disclosures and the companies I invest in or advise, please visit
02:50:04.380 peteratiamd.com forward slash about where I keep an up-to-date and active list of all disclosures.
02:50:28.220 Thank you.