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

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

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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 0.81

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 0.96

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 0.91

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 1.00

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. 0.99

01:47:08.320 Yeah. No, it's interesting. A little FK paste. 0.97

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 0.98

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, 0.99

02:29:12.760 but that's the challenge with designing trials. 0.98

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 1.00

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. 0.51

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 0.98

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

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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.

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