# We Spent $724,637 Testing Rapamycin. What We Found Shocked Us.

## Метаданные

- **Канал:** Dr Brad Stanfield
- **YouTube:** https://www.youtube.com/watch?v=Rcgiv8PoNBc
- **Дата:** 22.04.2026
- **Длительность:** 42:22
- **Просмотры:** 29,346
- **Источник:** https://ekstraktznaniy.ru/video/49355

## Описание

Exercise and Weekly Sirolimus (Rapamycin) in Older Adults: RAPA-EX-01 Randomised, Double-Blind, Placebo-Controlled Trial https://onlinelibrary.wiley.com/doi/10.1002/jcsm.70274

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Timestamps:
00:00 Background of the Rapamycin Study
05:44 Study Design and Protocol
07:33 - Results of the Rapamycin Study
09:29 Interpretation of the Results
26:28 Study Conclusion and Mechanistic Explanations
31:47 Discussion on the Impact of Rapamycin on Exercise
32:41 Discussion on Future Research and Dosing Frequency

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## Транскрипт

### Background of the Rapamycin Study []

We spent 5 years and $724,637 of crowdfunded money to test whether rapamycin would improve muscle performance when combined with exercise. And our results have just been published in the Journal of Cachexia, Sarcopenia, and Muscle. And they did floor me when we first calculated them. So, here to discuss the results is one of the co-authors of the study, Professor Matt Kaeberlein. So, Matt, you know the preclinical rapamycin research better than anyone. So, why are you excited about rapamycin's potential role for human health? And when I first contacted you all of those years ago, why did you want to be involved in this particular study? Sure. Well, first of all, Brad, thanks for having me on again to discuss the results of this study. You know, as you know, I've been studying the biology of aging for a couple of decades now and done some work on rapamycin in particular over that time. And you know, work from my lab and from many others in the field, you know, 5 years ago when you were first starting to think about this study, had really placed rapamycin as the gold standard for a drug that seemed to slow aging, increase lifespan, broadly improve healthspan in laboratory animals, and all the way from very simple organisms like yeast and worms up to rodents. And so, you know, from that perspective, it seemed to me to be very reasonable to want to ask, is there evidence that rapamycin can have an impact on healthspan metrics in people? At that time, I had also started on a series of clinical trials in companion dogs living with their owners in the real world testing rapamycin. And there, the results I would characterize as preliminary, but we're also looking positive for things like heart function, activity, and quality of life. So, we had a little bit of indication, first of all, that rapamycin could be tested safely in a clinical trial setting, albeit a veterinary clinical trial. And you know, at least directionally, it looked like certain healthspan metrics were improving in the context of aged dogs living in the real world. So, for that reason, you know, to me it's I still believe that rapamycin is our best shot on goal if we want to pick a single drug to look at in the context of biological aging in humans. And so, of course, it made sense to want to do what I could to help you get this clinical trial off the ground. And of course, I like to do cool stuff with cool people. You're a cool dude, so it seemed like a no-brainer for me to do what I could to help out. Yeah, thanks, Matt. And with the design of the study, you know, because some people have been asking why didn't we do a lifespan study? And if we did a lifespan study in humans for you know, that would take decades. So, you know, one of the reasons why we selected muscle is that if we could improve aspects of muscle function, that would significantly improve, you know, markers of frailty and that type of thing. And one of the things when I was looking through the rapamycin literature that caught me, and again, this informed the design of the study, is that it seems that in aged human muscles, we over activate mTOR complex 1. And you know, rapamycin, it blocks mTOR. So, one of the issues if you over activate mTOR complex 1 is that you don't allow another process called autophagy to happen. So, in theory, all of our aged muscles are holding onto these old damaged components and never flushing them away. So, that it was hoped that if we could cycle rapamycin, so have periods of time where we take rapamycin, we block mTOR complex 1, we can allow autophagy to happen. And then when we exercise again, we can switch mTOR back on and rebuild those new components. So, that was kind of the basis of the cycling hypothesis. And again, that's why we wanted to do a muscle study rather than a lifespan study. And yeah, the number So, when I first pitched this idea about 5 years ago, I was actually looking through the YouTube analytics about this. I didn't kind of realize how expensive these types of trials would be. So, yeah, it was a lot of the 5-year journey was actually trying to figure out ways to actually get that funding. But without further ado, is any other bits and pieces that you wanted to go through in the study before we actually go into the results? — I think one thing I would add to the comment you just made, actually two things. One is you know, I agree with your characterization that there's evidence for mTOR hyperactivation or at least suboptimal activation of mTOR in aging in muscle in particular. One of the consequences of that is impaired autophagy. There are other consequences including mistranslation of proteins, inefficient mitochondrial function. So, I just want to be careful not to suggest that autophagy is the only reason actually to think that rapamycin could be beneficial. And I also want to mention there's an additional line of evidence supporting looking at muscle function in aged people, which is that in rodents, it's a it's not an apples-to-apples comparison to what was done in this study, but there is evidence, I think better in rats than in mice, but even in mice, that in the context of normal aging, rapamycin can protect against muscle degeneration, sarcopenia, maintain body composition better. So, there was preclinical data to suggest that this was a reasonable healthspan metric to look at. And I think it's important to to put that out there because there is a perception that mTOR inhibition of mTOR is always going to be detrimental to muscle function because you because it's been well characterized for many, many years in exercise physiology that you need mTOR activation to build new muscle. And so, I just want to lay that groundwork to say, you know, this wasn't a crazy idea. There were plenty of reasons to think that this was a logical thing to measure as an endpoint or series of endpoints in a human clinical trial. In

### Study Design and Protocol [5:44]

terms of how we actually designed the study, what we wanted to do is try and make sure that the protocol that we were giving patients was actually going to be what patients could do in the real world. So, what we did is we took 40 participants. Half of them took sirolimus or rapamycin 6 mg and the other half took placebo, but both groups were exercising. And the exercise protocol was that we actually delivered exercise bikes to their homes because these were older adults aged between 65 to 85. And again, we wanted to make sure that this exercise program was going to be something that they could easily do within their homes that was safe as well because, you know, the last thing we wanted is for an 85-year-old to do, you know, high-intensity interval training and it cause some sort of injury. So, that's why we set this study up in the way that we did. And it was it ran for 12 weeks. So, that's I think also important is that it's a relatively short-term study. And the And people were taking either the rapamycin or placebo on day six. So, they would exercise on day one, day three, and then day five. And then they would take rapamycin on day six. And then again, they did that for 12 weeks. And the hope was that if we would if we dosed the rapamycin on Saturday, in theory, most of the rapamycin should be out of their system by the time that they start exercising again on Monday. So, that was the overall gist. And So, before I go into that, one of the things that we that I really wanted to make sure about when we set up the study is that we wanted to we wanted to have all of our endpoints registered. So, what I mean by this is that we actually were on a clinical trial database so that people could know exactly what the study was doing, what it was measuring. So, you couldn't fudge anything. There's no p-value hacking or anything like that. And you can follow exactly what we did. And so, all of these endpoints and all of the sensitivity analyses, they were prespecified. So, yeah, again, no fudging of numbers. So, the primary

### Results of the Rapamycin Study [7:33]

outcome, so the one thing that we wanted to test that the main was the 30-second chair stand test. So, this is how many times can you stand up and then sit back down in 30 seconds? And we chose that marker because it's relatively sensitive for overall muscle power and strength in older adults. Again, we had 40 participants. And the primary intention-to-treat analysis, so this means that you're including everyone in the study. This one didn't reach statistical significance, but it did trend towards the placebo group actually improving their 30-second chair stand test more compared to the placebo group, which is a interesting finding, but again, it's not statistically significant. So, I don't think we should necessarily over interpret this. But then when we did have a look at the sensitivity analysis, and again, all of this is pre-specified, that's why it's here. Um, when we just looked at the people that completed, so it's called case complete. So, these are the people that we managed to collect data on. That is when we could see statistically significant results where the placebo group was improving their scores more compared to the rapamycin group. And then if we further drill down, if we have a look at the per protocol analysis, so this is people defined as they either took 75% or more of the study drug, and they did exercise plan. So, these were the people that really stuck to the protocol. This is where the difference in the starts to widen. And again, it's more Well, that's a misnomer, but the Cohen's D or the effect size is greater. So, again, it seems that the more that people were sticking to this protocol, the more divergence there was between these two arms, which is a it's not exactly the results that we had predicted going into the study. So, I think I'll stop there. Matt, when you first, you know, came across these results when we calculated them together, what were kind of your initial impressions? Sure. So

### Interpretation of the Results [9:29]

a couple things I would say up front. So, first of all, I want to emphasize what you said, which is that all of this was prespecified. I really think this clinical trial was done to the very highest standards you could really possibly do a clinical trial to in terms of rigor. People can criticize the sample size and the length and duration of the study and all of that. But in terms of the design and really trying to take a rigorous unbiased approach, I think you did everything that that could be asked should be asked. And so that really provides confidence in at least the interpretation, right, of the outcome. And so there's two pieces here that I think are really important. So one is it did not hit statistically statistical significance for the primary endpoint. And that's the most statistically rigorous analysis. Even though the sensitivity analysis was pre-specified, it is a less robust type of analysis that breaks randomization. So I think it's just important for people to appreciate that. It's not P value hacking, but it is predisposed to false positive results. In this case, the false positive potentially. I'm not suggesting it is really a false positive potentially being the statistically significant difference that was observed. That's just the nature of doing these sorts of additional types of analyses, especially sensitivity analysis that breaks the randomization. It is intentionally designed to be more susceptible to false positives with the expectation that there will be future trials that can really determine was it a true positive or a false positive. So for that reason, I think it I personally have less confidence. And I'll give you my like take home, what do I actually believe? But just from a pure statistical perspective, I have less confidence that there is a real statistical difference between rapamycin and the placebo group because you only see that in the sensitivity analysis. Having said that, the other thing I think that's important to appreciate is regardless of whether that's a true difference, both groups showed improvement. The one interpretation, which is really where I land what in terms of what I believe in my heart, is that the placebo group improved more than the rapamycin group. Or another way to say that is rapamycin attenuated the improvement in function in the chair stand test, right? But both groups showed improvement, at least directionally, whether that's statistically significant or not directionally. So that's kind of my That's what I think happened here, right? And again, there's what can we say with 100% confidence based on the the the Maybe I should say 95% confidence cuz that's the typical P value confidence threshold. Versus what do I actually think is going on? So I think given the exercise regimen, the dosing protocol, the length of the study, all of the factors that were involved in the study design, rapamycin attenuated the improvements in muscle function based on these tests in this population, but both groups improved. And I think the other thing to say is if you look at panel B there, right? I think this can help people appreciate how noisy human data is, right? This is why it is hard to see statistical differences, you know, even with 100 people in each group because there's so much individual variation. Many of the rapamycin users improved as much as the placebo users. Some of the placebo group actually went down in function. It's all over the place. And so that's why we really need to do, you know, well-powered clinical trials for when we're talking about human studies, especially in healthy people where we're not really trying to target a specific pathology of a disease. I don't know. Does that align more or less with kind of the way you think about this or do you have different thoughts on the outcomes? Yeah, I'd say mostly. I think it's critical that we don't necessarily over-interpret these results. The other thing that I probably would say just skipping here to the secondary outcomes because the other measures of muscle performance that we used were the 6-minute walk test as well as hand grip strength. And those ones didn't reach statistical significance, but they still trended in the direction that the placebo group improved more compared to the rapamycin group. But again, I agree with your overall point that we mustn't over-interpret these results, particularly since the the main you know, primary endpoint wasn't statistically significant. But I do think that it starts to paint a bit of a picture that over a 12-week period dosing once a week, at least in this population may maybe you know, either too much or they need some sort of a washout period cuz that's the thing it would be quite it would be really interesting to have repeated this if sorry, if we stopped the study drugs at the 12-week mark, but then continued the both groups exercising and then tested them again at the 24-week mark because you might find that the curves reverse. But again, I I think that over at least a 12-week period in this population I think this data starts to paint a a bit of a picture that you know, may maybe the dosing protocol might not be optimal. Yeah, I mean I think that's a very reasonable hypothesis, right? And and so obviously, you know, in a perfect world, we'd be able to test that hypothesis. Hopefully we will or someone else will be able to. One of a couple other things I want to mention because you know, that have occurred to me since we first started looking at these results as well. I mean I think what you said about the fact that even in all the secondary analyses, even though they didn't reach statistical significance, directionally, they're all suggesting that the rapamycin group did not perform as well as the placebo group or didn't see as large of improvements as the placebo group. When you start to see directional changes across multiple endpoints in a clinical trial, that even though statistically we can't say that improves our confidence about the statistical significance, it should appropriately make you consider that it is probably likely that that's a real result. At least again, that's the way that I view this through my sort of pragmatic lens that it seems as though across many, if not all of the outcomes that were being assessed in this clinical trial, the rapamycin group did not perform as well as the placebo group, right? And so when you see that directionality over and over, you start to get confidence, okay, this is probably a real signal. We don't necessarily know what the explanation is. The other thing though that I find a little bit surprising, and this is where I think putting it in context of the data that we have, you know, across many Maybe not many many, across many different studies, right? And let's leave the preclinical stuff out of it and just think about the human studies with rapamycin. This is really the first study I can think of where it really seems to be a pretty clear signal that rapamycin underperformed the placebo. So that's a little bit surprising, and we don't have very many in the context of muscle function in humans. But you know, there was the Pearl trial, and that's got a lot of challenges in interpretation as well. But over a larger period, I think it was 48 weeks or something like that, a longer period, at least in women, there did seem to be a change positive change in body composition in the rapamycin group. Now, body composition is different from, you know, sit stand test or 6-minute walk. So it could be the way it was measured. But that's at least a a different clinical trial that got an apparent opposite outcome. And that again may be because that was a longer time frame. Dosing was different. We can talk about dosing cuz I think that's that's a leading hypothesis here is that this dose was probably higher than optimal, at least for these particular outcomes over this time frame. The Pearl trial attempted to do 5 migs and 10 migs once a week, but because of formulation challenges, they used compounded rapamycin without an enteric coating. Estimates are that their effective dose was around 1, 1 and 1/2 migs per week. So if that's correct, that was a lower effective dose than this study and a longer time frame and a different patient population. So, you know, this I think you appreciate now many of the things that I learned when I was designing the clinical trials in dogs. When you're designing a clinical trial, you have to make a series of decisions, best guesses about all of the different parameters that go into the clinical trial. And then you get to the end and you're like, oh boy, maybe we should have done it differently. But you do the best you can with the information you've got at the outset. Yeah, no, I completely agree with that. And just to finish up the results section, so you know, we measured a few other things like the SF-36 questionnaire. So this is just a questionnaire looking at different health-related metrics. There was no statistically significant results there. We had a look at CRP, which is a marker of inflammation. Now, with this, I did just want to spend a bit of time on this because you'd expect that with rapamycin, that should overall lower inflammation. Whereas we saw the opposite, but this was a bit of noise in the data because there were two outliers in the treatment group. And they had both marked elevations of CRP. And CRP can go up for a variety of different reasons. If you've got, you know, a cough or a cold at the time, if you've banged your knee, if you've you know, done something like that, CRP can go up. So I don't think that you know, people should over-interpret the CRP rise within the sirolimus arm because again, I think that's just noise. We had a look at, you know, safety and adverse events. Overall, there were a higher number of adverse events in the sirolimus arm compared to the placebo arm. But most of these were just they were to do with, you know, muscle aches and pains from, you know, from the exercise protocol. So again, I don't think that we should necessarily over-interpret this. The one thing that is of note is that there was a hospitalization that occurred when we were doing the study. So there was one particular patient who took one dose of sirolimus. And I think a few days later, they ended up in hospital with pneumonia. Now, it's a bit unclear looking through the records whether the person was actually feeling unwell before they started taking the study drug or not. But again, it just important to note. And since this is such a small study, again, I don't think that people should be necessarily over interpreting this. Uh Yeah. Let me just For people who may not be rapamycin aficionados, all right? The reason that's suggestive, of course, is because rapamycin is used in organ transplant patients as an immunosuppressant. You would expect if there's going to be one likely type of side effect from off-label use of rapamycin, it would be an increased risk, particularly of that bacterial infection. So, it's suggestive, certainly, and appropriately, you know, was judged to be potentially due to the study drug. And that's it, I think, for the results. Um, that you know, we looked at other lab uh markers, and in particular, we were looking at things like HbA1c, um, which is a marker um of your last 3 months uh blood sugar levels, and there was a slight increase in um in the sirolimus group compared to placebo that did reach statistical significance, but that the clinical difference is so small that to me, if I was looking at this as a um as a GP, you know, uh and sorry, these are in New Zealand or UK units. The difference is so small that it to me, it doesn't actually have a clinical relevance to the patient's um health. So, yeah, I was actually quite um relieved when I saw these uh safety uh lab results. Um Matt, did you have any sort of comments about these? Yeah, I was just going to say, again, I think, um, you know, uh gluco-regulatory homeostasis is one of the common side effects in organ heart transplant patients that is uh perturbed by rapamycin. So, if again, we were going to see side effects, we would expect it to be lipids or things like A1C, insulin sensitivity. Again, this is suggestive that there probably were modest effects of rapamycin in this population on glucose homeostasis, potentially insulin sensitivity. Um so, again, I view this I view the entire trial as sort of moderately cautionary. And one of the things again that surprises me is there's other data out there, some in larger populations, not as well controlled as this study, on rapamycin at comparable doses or using everolimus in the case of Joan Maddox study, at comparable doses, where you really didn't see this increase in side effects. And so, it makes me wonder if, um, you know, this was a particular population where it seems that this dose of rapamycin might have been too high, or if that Rapamune was used in this case. Most of the other studies use a generic form of sirolimus. Is there differences in bioavailability there? But it Again, if I had to guess, and I hate I I like guessing, I just wish I knew with more certainty um about my guess. Uh if I had to guess, the dose was just a little high, and maybe spreading it out um longer, so it wasn't once weekly, would have alleviated that, and we wouldn't have seen, you know, this moderately or modestly concerning signal when it comes to safety. The the cheap way, I suppose, of getting uh sirolimus is a generic version, but the trouble with some of the compounded generic versions is that, yeah, they aren't enteric coated, and they can just be broken down uh by the stomach acid and not properly absorbed. Um so, we wanted to make sure that this trial didn't have any issues like that. So, we purchased um sirolimus directly from Pfizer, and Pfizer had no role to play in the study. They didn't even donate their study drug to us. We had to pay them to have the study drug. The other issue that we uh then encountered is that So, you can't crack open uh these capsules, because if you do, then again, the stomach acid will just get into uh those capsules, break them down, and you won't actually absorb uh the rapamycin. So, what we had to do Um and sorry, there's also no matching placebo. So, what we had to do is we had to get these triangle capsules, and uh what sorry, tablets, and we had to put them into larger capsules, and then fill the gaps with um with cellulose. Uh and then we had the matching uh placebo as the uh as the same capsule, but that one was just filled with um with cellulose. So, that might be a potential reason as to why we saw these results is that, you know, by using sirolimus from Pfizer, we made sure that the 6 mg dose, people were actually truly having that 6 mg dose. That's right. Yeah, and again, it's again, this is getting in the weeds a little bit, but it's worth just noting that, you know, Rapamune is sort of the brand name generic. Let me just put it that way, right? So, that I think most That That's the version of the drug that when somebody makes an makes a different generic, so you can get just sirolimus from Dr. Reddy's or other manufacturers that isn't Rapamune, in theory, those generics are supposed to give you the same pharmacokinetics and bioavailability as Rapamune, but they're formulated differently, and I don't actually know whether or not they are identical in terms of bioavailability, but if anything, they would be lower. And then, as you alluded to, compounding pharmacies um often use their own packaging materials. Some of them use an enteric coating, which should preserve bioavailability, but we have no idea whether it 100% preserves bioavailability. And then, like in the case of the Pearl trial, some compounding pharmacies don't use enteric coatings, and we know in that case, 70 or so percent of the rapamycin does not make it into circulation. So, there And this is where again, if we could go back, you know, had more resources from the very beginning, we would have loved to measure rapamycin levels in blood, you know, multiple times during the course of the study to actually be able to assess bioavailability, clearance, all of that stuff. Um but we just don't unfortunately don't have that data to be able to know what the pharmacokinetics looks like. Um I will say, I mean, I think I'm guessing you'll want to come back to this idea of, you know, longer spacing between time points. Um I just did an uh an interview with Bonnie LeFleur at the University of Arizona. They're running a large clinical trial down there, and they've done a preliminary study on 50 people um taking either four or eight migs per week. And um and so, we'll start to get and they do have pharmacokinetic data on at least those 50 people. We'll start to get more data on what the pharmacokinetics of this once-weekly dosing of rapamycin looks like across a heterogeneous population. My guess is, you know, much like the data um that we looked at from this paper, it's going to be very variable in individuals um the rate at which rapamycin is cleared, at least from blood or from serum. Which kind of brings us onto

### Study Conclusion and Mechanistic Explanations [26:28]

the discussion uh section of the study. So, um again, so I'm just going to read this, cuz I think it's important. Contrary to the cycling hypothesis, so this again, that's the hypothesis where we were hoping to have periods of time where we're switching mTOR off by taking rapamycin, and then separate periods of time where we're trying to switch mTOR on by uh through exercise. Can I just add one thing there? I would frame it as we're trying to turn down mTOR, not off. I just so cuz I think too often people think I know you know, but a lot of people think it's on or off. It's a volume dial. Yeah. Correct. Yeah. Um so, in the study, we found no evidence of benefit. Instead, this the sensitivity analysis revealed statistically significant blunting of functional gains in the sirolimus arm. And again, I um I do want to preface that and agree with what you're saying, Matt, that we mustn't necessarily over interpret that. Um we've talked about, you know, some of the context and comparison with previous studies, in particular, the Pearl study. Um what I sort of wanted to talk about is a bit of the mechanistic explanations. So, uh in terms of why we saw these results. So, in the paper, we said the failure to achieve a best-of-both-worlds effect, which it um which again, was trying to have um separate period Well, we've just talked about this, but the cycling hypothesis, where we're switching down mTOR, um and then switching it up uh with exercise. I don't think we quite nailed that um that balance in the study. So, even though we dosed sirolimus 24 hours after the final weekly exercise session. So, remember, people were exercising on day one, day three, day five, and then they were taking the sirolimus. Um you know, in the data that we do have, and Matt, maybe you want to talk about this, but the half-life of sirolimus is roughly uh 62 hours, um which may have meant that there was some uh sirolimus still within the system within the body system uh during, you know, the first or even second exercise session. So, on day one and day three. Now, Matt, I know that you've um got some ideas and thoughts around that half-life number there. So, did you want to sort of expand on that? Sure. Yeah, well, I think the way I would uh describe it is most of the data we have on pharmacokinetics of rapamycin come from studies of organ transplant patients. There are other patient populations where rapamycin has been used, but the vast majority of data comes from organ transplant patients who are taking several milligrams every day, right? And so, in that context, what happens is you rapidly build up uh steady state level of rapamycin in the blood. And so, then what you're actually measuring with this 62-hour terminal half-life is the rate of metabolism of rapamycin uh through cytochrome P450 systems and probably other systems that break the drug down. Um but that's a different context than what we're looking at here. And presumably, those cytochrome P450 detoxification systems become sort of maximally overwhelmed when you have a high level of rapamycin in the system. That's a little bit speculative. So, all I would say is I'm a little bit hesitant to say that the half-life of rapamycin through that particular pathway in people dosing once a week is actually 62 hours, and also we're saying there's interindividual variability. What we have some data on are what is the level of rapamycin in serum in people dosing once a week. And again, the data is not published yet, but Bonnie shared it with me recently. In their study, again, 50 people, 4 migs and 8 migs, essentially everyone, maybe with the exception of one person, were getting below the detection limit before the next dose on that once weekly dosing schedule in serum, okay? And they were not seeing accumulation in serum over the course of the study. So, what that tells me is, at least looking at the serum levels, most people are clearing the drug in that week before the next dose. And the fact that you don't see accumulation of the drug over an 8- or 12-week period suggests that in fact that fits with the idea that they're actually clearing it. Now, the caveat I would add to that is, when you're measuring in serum, that doesn't actually tell you what's happening in muscle cells or liver cells or kidney cells. So, it is likely that there is still rapamycin present in muscle, in liver, in kidney, and in other tissues inside the cells that could be inhibiting mTOR even if we can't detect it in serum. So, I think we don't unfortunately, without doing a muscle biopsy and measuring mTOR signaling, we can't really know for sure whether mTOR complex 1 was in fact inhibited in the muscle of these individuals or in other tissues, you know, 3, 4, 5, 6 days after their most recent dose. I think your hypothesis is very reasonable, likely to be correct. That could account for why there appeared to be an attenuation of the improvements in these individuals. Yeah, a couple of things with that new study that you're quoting. Do you know if they use sirolimus from Pfizer? I don't know the source. I know it was not compounded, so it was either Rapamune or a generic sirolimus that had enteric capsulation built into the formulation. Okay. And then the other thing I suppose

### Discussion on the Impact of Rapamycin on Exercise [31:47]

is that even if the yeah, as you were saying, they it seems likely that most if not all of the sirolimus is out of the system with before the next weekly dose, but we don't really know what happens at day two or day three. And if there's still sirolimus within the system at day two or day three, then that would have potentially impacted the benefits of exercise on that day one exercise day. Yeah. No, I think that's right. And and we do have some data on that. And in fact, this is where the interindividual variability seems interesting. From the data I've seen, yes, most people will still have detectable levels of rapamycin. It's pretty low, but you can still detect it over 1 ng/mL at day two, sometimes at day three. So, yes, it is very likely that there was some degree of mTOR complex 1 inhibition in those people on at least the first day of exercise. And that's why in implications for future research

### Discussion on Future Research and Dosing Frequency [32:41]

in the in the study that we published, we said future research should explore ideas that lengthen the time between doses, for example, every 3 weeks to every 6 weeks. Um And again, this is to try and make sure that all of this all of the sirolimus is out of the system by the time that you're doing most of your exercise days. Um and so, that's sort of the next study that I would love to be able to run. The trouble is, to do such a study, ideally you'd want to do a 12-month study, and you'd probably want to have a three-arm study. So, you'd placebo arm, one dose every 3 weeks, and then one dose every 6 weeks. Um so, to do such a study, you know, you're probably looking at a multi-million dollar study, realistically. Um and yeah, so to try and find funding for that is going to be challenging, to say the least. Yeah, I I would agree with that. It is challenging, for sure. Um I do think there are some other hypotheses that I are worth addressing as well. So, um you know, I wonder whether or not uh if there'd been a washout period for 12 weeks, whether the rapamycin group would have caught up. And so, this is where I think the design of the study and what you're measuring is really important to consider. This is Correct me if you feel differently, but in my view, this study is really looking at people who have not been on a dedicated exercise training regimen and putting them on one, right? Um and yes, that's what's happening in the real world. So, so I I fully recognize that. But, those people are going to see pretty rapid They should see improvements. Most people should see improvements. That's potentially a different outcome than in people who have been exercising for a while, and you're looking at the ability of rapamycin to improve sort of homeostasis within muscle over the longer period. I wonder if in a different population, this design or design where they got 12 weeks once a week and then 12 weeks without rapamycin, and they continued their normal exercise regimen, if it would have shown a different outcome. If rapamycin might have been the same, might have even been better in that context. So, I wish we could test these different variations. And I think the take home for people to understand is, because there hasn't been resources to do the kinds of clinical trials that you've been advocating for that I've been advocating for, when it comes to dosing and frequency of dosing, we're kind of stumbling around in the dark. We've got a little bit of evidence. Some of it's cultural, some of it's based on hypotheses like the idea that once weekly will avoid off-target inhibition of mTOR complex 1. Um but we don't actually have good quality data to tell us whether or not is 1 week better than 2 weeks is better than 3 weeks uh breaks between doses. We just don't have that data. So, we're guessing in many ways um in the absence of having high high-quality data. The last thing I'll say as well is, the optimal dose for brain or heart may be different than the optimal dose for muscle. And uh again, we don't really have enough data to be able to to I think even have, you know, real confidence in our guess as to whether that that's going to be the case. But, it wouldn't surprise me if, you know, to get some of the potential benefits in brain, for which there's accumulating evidence, you need to go to higher doses. But, that may offset some of the benefits you're going to get in muscle, if there are going to be any benefits from rapamycin in muscle. Yeah, a couple of things. So, we we purposely chose in the selection criteria or entry criteria to the study, we purposely chose people that didn't already do lots of exercise. And the reason for that is we did want to see a change in the 30-second chair stand test, you know, with the before and after. But, also I was hoping to see that by using rapamycin once a week, we might actually help treat the age-related anabolic resistance. As in, you know, some an 80-year-old who exercises is going to have less benefits of exercise compared to say a 20-year-old. As in, that 20-year-old would you know, improve in muscle strength and power and speed quicker compared to an 80-year-old who's doing the same exercise protocol. So, I was hoping that if we managed to get the dose right and if the cycling hypothesis worked, that we would be able to see not only that the rapamycin group improved. Well, I was hoping to see that the rapamycin group would actually improve more compared to the placebo group because of that potential effect. Um and even if you take sort of step forward to that washout period, if you need a washout period, to me that still indicates that the once weekly dose is too much. And I think that that's sort of the to me, that's the central takeaway of this. I think once a week dose, at least for older adults in the context of muscle performance, is too frequent. And that if I think we you'd be more likely to have a positive outcome if you dose every 3 weeks, every 6 weeks. And I think that once a week dosing protocol that we initially chose for the study, as you say, we are kind of guessing. And I think a lot of that was just cultural that at the time, a lot of people within, you know, the so-called longevity space were taking rapamycin once a week. Um I agree, although there is data from Joan's studies at least to suggest that when it comes to vaccine response, once weekly is as effective as daily and has a lower side effect profile. So, there is some clinical trial data to support that. But, yes, it is largely cultural. Um let me give a little bit of an alternative hypothesis. And again, we just have to be honest, we're hypothesizing. We don't know, right? But, but um part of the reason why I think there's at least a possibility that a period of relatively high inhibition of mTOR complex 1 given once weekly, so that you can avoid some of the metabolic side effects potentially, um may be useful for certain endpoints is because with age, we have this dramatic increase in chronic inflammation, right? I think everybody would agree with that across multiple tissues and organs. And that chronic inflammatory state impairs the normal function of those tissues and organs and stem cells and other cells within those tissues and organs. And from the preclinical data in mammals, and this is mostly rodents, it seems as though you need between 4 and 8 weeks of pretty consistent relatively high mTOR complex 1 inhibition to bring down that chronic inflammatory state. And then when you stop the treatment, you often see robust improvement in function because now that the stem cells, presumably, this is a little bit speculative, or the immune system can function more like a youthful system because you blunted that chronic inflammation. So, it is possible that a 4 to 8-week period of relatively robust mTOR complex 1 inhibition followed by a period where you allow return of normal mTOR homeostasis, you need that to get some of the benefits that we see in preclinical models from rapamycin. And again, this is where the endpoint probably matters. If it's a hypertrophic endpoint, you may not need that. But if it's more of an immune-mediated endpoint, you might need that sort of, you know, washout period to actually see the the response. So, again, I think it really is going to We just don't have a good enough understanding of really even the biological mechanisms that are underlying many of the benefits of rapamycin even in the preclinical models. So, we're guided by, you know, our best models that we've got right now. But, you know, those models may be incomplete or incorrect. Yeah. Yeah, no, I'd agree with that. And it's just such a shame that we don't have enough funding in this field to be able to, you know, answer these questions. Um you know, I'd love to be able to do a follow-up study that can hopefully um yeah, move us closer to the truth. Um Yes. but again, 100% to do such to do the next phase of the study I believe me, I I've been around long enough to have experienced uh many many many frustrations with how challenging it is to get funding. And um you know, the studies we will hope will eventually get done. I'm optimistic. I think this study out of the University of Arizona not designed specifically for muscle function, but that will answer some important questions. I'm uh enthusiastic that it it's a relatively large study, 700-plus people. So, you know, my uh frustrated side of my personality is like, "Yeah, but that should have been done 10 years ago. " Nonetheless, it seems as though it's going to be done now, and um hopefully going forward, you know, we'll be able to join forces again to do, you know, answer the kinds of questions that we have uh brought up on this uh this conversation today. And man, I really uh wanted to thank you for all of your support during this journey cuz it's been Yeah, a 5-year 6-year journey. Um and I've just, you know, really appreciated being able to bounce ideas um and have these types of conversations with you. So, thank you. Right back at you, man. And I have so much respect for you for your persistence and pushing this forward and being able to put some of your own, you know, personal resources on the line to do this clinical trial. You are truly dedicated. Uh you know, I just really value you as a friend and want to thank you for being who you are and for doing the right thing. Thanks, man.