A transcription of the interview / discussion:
I used otter.ai for this transcription and have cleaned it up a bit but haven’t gone through it too carefully so there may be my mistakes and typos.
Speaker 1 - Adam Salmon
I think having a perspective from a folks who don’t have a biology background is also important too, right? I mean, not only for their perspective on the things that we’re putting out, but coming up with new ideas, right? Why not this approach? And I see some of the questions here, you know, they’re insightful, the ones we sent, they’re very insightful. And I think we don’t always have in our minds that direct, translational sort of aspect as basic biologists. So it’s great to take ideas around with other folks. Oh, great, super
Speaker 2 0:31
I appreciate that. So you’ve probably done a great deal of studying on mTOR. I assume, right, you understand that pretty well in depth.
Speaker 1 0:39
well. I would say my background is more on rapamycin as an intervention and less on the, you know, the functional biological molecular effects of informed on, you know, I’m pretty well versed in it, but we’re not really following up on, you know, this molecule does this, this does that. It’s really, what benefit can we have on health and aging? And you’ve been working on this for how long now? So this particular project, we were initially funded in 2015 really got started going in 2016 with enrolling animals. So now six years, seven years, and it’s a long project, right? You were talking about primaries. We’re not talking about mice, and some of it’s just taking the time to our animal saved?
Speaker 2 1:22
You do you have a good understanding of when mTOR starts becoming dysregulated, or, you know what period in the lifespan? Is it around? in mice, they talk about they started doing the first ITP test at around nine months?
Speaker 1 1:36
I think it’s not really clear, right? If you did venture to me, no one’s really said here’s a time dysregulated, or that with age, it’s always dysregulated in every tissue, right? There’s, there’s sort of this assumption from, you know, maybe from invertebrates, maybe from mice, and it’s just like a progression, and it’s sort of the same everywhere. But I don’t think it’s clear that it is okay. And a lot of the stuff that folks have been looking at is kind of like steady state, right? Okay? What it looks like in a Fed animal or a fasted animal. This is a responsive system. And rule we really need to be looking at are sort of challenges, and what happens after the fact that’s that’s where the money is. It’s my my knowledge, that’s not clear at all.
Speaker 2 2:22
Okay, do we? Is there a way to measure mTOR right now?
Speaker 1 2:27
so, I mean, we use surrogate markers, mostly, you know, phosphorylation of proteins in the mTOR signaling pathway, uh, upregulation of different downstream you can even measure the how, how the infor complex is forming or not forming in certain scenarios. But the problem still comes back to in tissues, in animals and people, it’s always sort of a static look, right at this particular point, that’s what it looks like. We’ve got a little better way to modulate them and look at response and sort of kinetics of that response, but cells aren’t a great model of humans, and
Speaker 2 3:04
we also don’t have much visibility in terms of tissue specificity or anything like that, really, right?
Speaker 1 3:12
it’s very clear that, you know, import is differently regulated in different tissues, not in terms of what exit, but sort of the response and dosing even rapamycin itself. In mice, there are different responses in different tissues on their makeup.
Speaker 2 3:31
One of the things that we’ve wondered a lot about is, are there potentially different delivery mechanisms that will affect the tissue specificity of rapamycin if you did subcutaneous injection or intranasal delivery. Is that going to affect the distribution of mTOR inhibition?
Speaker 1 3:58
I mean, I think there’s no question my mind. I don’t think anybody’s really tested it beyond maybe some mouse work where they’ve done sort of in the diet, like the ITP has gone versus an injection intraperitoneally, and you can see there are differences in that response, both time, magnitude, et cetera. But to be honest, like a systematic weather, intranasal, transdermal, anything like that might, might have similar effects. I don’t think it’s clear, but I think we’re pretty confident it will have to, would
Speaker 2 4:29
probably be an interesting series of studies to be done. I assume
Speaker 1 4:34
Absolutely, and that’s thing, I think, not just with rapamycin, but all of Aging studies, we tend to make assumption based on data that’s been published that but we don’t make comparisons, and it’s hard because you have lots of groups and lots of resources required, but so a lot of the stuff, we’re still at the very early stages. I mean, to be fair, human drugs, we can’t ever do the same thing when
Speaker 2 4:58
we say. Rapamycin is a very good mTOR one inhibitor. What does that mean? Specifically? I know that it blocks that production of proteins very well. How long does it last? Is there a standard degradation curve over time? How is it impacted by food or other traditional mTOR stimulators, once you, once you blocked it. Does that mean it’s blocked for a certain amount of time? Or does eating protein, or, you know, light elucine Or or or other traditional activators, do they unblock it? Yeah,
Speaker 1 5:37
so I’m going to speak from my experience in practical application, the experience in the pharmacokinetics and pharmacology that somebody else might might have a different okay, what, what we see with application. And most of my so I take it back. Most of my work is still in mouse models. It’s all one sort of extension. So I’m really a mouse and basic biologists. Well, what we see is that with that chronic rapamycin administration, the diet, where they can be constantly they there is basically a blocking of mtors or blunting of mTOR response to other stimuli. So for instance, glucose and normally I’m sorry, you get a big response, because this transient pathway get as a very blunted response when rapamycin, it doesn’t take very long mouse models for that to once you remove rapamycin, within weeks, they’re back to a normal sort of response to that. Okay, permanent shift, it likely is, as long as the rapamycin is available, it’s it’s causing this, because it’s binding here in the washing and once you reach the half life of the drug, I think essentially, it’s going to go back. So there may be some, some conditional issues,
Speaker 2
one half life.
Speaker 1
I mean, probably more than that, because even one half by half the amount is going to be around, and it’s going to slowly return back, you know. So that’s the direct effects on Mtor. But I think the indirect effects, if it had on epigenetics, well, because it boosted autophagy. Now you’ve cleared out bad proteins, those will certainly remain until all that drove it are back. That’s a whole nother conversation. Is the remaining effect after. And I know there have been studies by Matt caberlin group and other groups that have done the short term burst and then.
Speaker 1 7:34
And, you know, I mean, I think there’s good reason to believe that it’s the indirect effects, the downstream effects, that we’re really concerned about, not the direct effects on mTOR at the time, but we’ve really separated those out scientifically for no for sure. So
Speaker 2
mTOR inhibition doesn’t really have any physiological effect on the uptake of the nutrients in the body. So if you’re, if you’re in a mouse or a monkey, and you feed it protein while it’s while it’s suing rapamycin, it still gains the benefits of those nutrients. Is that correct?
Speaker 1
So I think probably not completely part, because rapamycin mTOR is a significant regulator of stem cells in our gut, right? So if chronic rapamycin affects that in a different way, I mean, in theory, it could affect uptake of different Okay, I think this is one of those things where nobody really tested it. What I know people have done is looked at things like, does it affect the microbiome? But they, I don’t know of anyone who’s gone and looked okay in the feces of a mouse on rapamycin. Is it the same amount of nutrients that have just passed through? I don’t know. You know, the same thing could be said. You’ve now completely modified the system if all the employers is inhibited inside uptake of nutrients at any stage in the way could, could be a better Okay, clear at this point.
Speaker 2 9:02
And you mentioned the microbiome, obviously, you see in mice that there is a significant impact, just by the size of the stools. What is, do you have any insights into what the effect is on microbiome? There hasn’t been a lot of research on that yet. It seems like
Speaker 1 9:16
no, I don’t know. And it could be twofold, right? It could be infor specific, on your gut for itself, is also an anti fungal and microbial just completely shifting your flora in your gut. That I don’t know, that we don’t conservative with my answers, because that’s the kind of I think scientist I am, and there’s a lot of stuff we don’t know. Rapha, do
Speaker 2 9:42
you have any feeling or opinions on whether the net effects of rapamycin on the microbiome are positive, or is it negative and it’s being overwhelmed by the positive effects in other areas?
Speaker 1 9:54
Yeah, I don’t know. Because in part of it, I don’t know that it’s. Still hard for me to discern. What are positive effects on changes in the microbiome beyond, okay, it’s associated with good health. Limited Data that changing the microbiome directly does these things. And I mean, I think it’s a growing field, but there’s limited evidence there, right?
Speaker 2 10:16
Yeah, I think in general, the microbiome field is really early, and even identifying a positive microbiome is still pretty undetermined. I think, right, yeah,
Speaker 1 10:24
and, and, I think it’s a whole different world too. We’re talking about a mouse microbiome versus application to human I mean, we eat different things. We don’t then really eat our feces in our cage like mice do. Kind of hard to see the translational aspect at a high level.
Speaker 2 10:39
How do you how do you think the marmoset studies compare to the dog studies in terms of translatability to humans? Do you, do you feel that they are better? I would expect them might perhaps be better, because obviously monkeys are a lot closer than then dogs are evolutionarily. But what’s your opinion?
Speaker 1 10:59
I don’t know about better. I think, I think I think they’re different, and we’re going to be able to glean things from both of them, right? I mean, for the reasons like you said, I think the primate, genetically, physiologically, socially, is a lot. It very similar to human sit down. There are downsides that we have to write. They have their own peculiarity. They we keep them in a lab state the dog study is going to be have that environmental component, which I think is an important thing. Ultimately, both are just a step closer to human application and just build us a little bit more than mouse or invertebrate studies. And I think they all need to be addressed. Ultimately, we’re modeling humans, and they’re still modest downfall.
Speaker 2 11:42
Okay, let me jump into some of the your specific papers that I read. So you monitor, how often do you do blood draws on the monkeys? And do you check our periodic basis? Are you monitoring the blood levels over time of different constituent, blood components, or less. So.
Speaker 1 12:02
So about every six months, we do a physical, physical on them. They get their blood work done, CBCs, lipids. But then we also, every month, bank blood. So we’re looking at that aren’t specifically towards general health. You know, part of this is viewing it like a clinical trial. We’re looking at their health on part of it is banking things for actual scientific
Speaker 2 12:24
Okay, do you have any sense on longevity purposes, whether it’s AUC?, CMax?, what do you think are key factors in in the effectiveness of rapamycin?
Speaker 1 12:36
So, you know, you you raise this point, I’ll have to come back to the mouse, because we don’t know. My general feeling is it’s probably something like the AUC and this is based on the fact that, you know, despite what some people might say, the chronic rapamycin treatment has still been the most consistent effect. And the one test that really go did it same time. Test different ways to do it, by the ITP every day in the diet, still did the best. So that my feeling is that I’m not saying that, that we’ve discovered this is the best dose, this is the best way to do it, right when you when those mice consistently have rapamycin in their blood at pharmacologically active level, they live the longer. So that’s, that’s my feeling now, there’s, there’s the counter to that is that these mice may be living with different diseases, that because they’re mice, they don’t die, right? Diabetes or heart disease, heart disease, and we don’t know that. Hopefully we’ll know more with mom and study because they do get they do get cardiovascular
Speaker 2 13:44
I don’t know if you saw the gladyschev Lab rapamycin study earlier, and they did on newborn mice, which was quite interesting. They saw a very dose dependent and significant reduction in growth size to the animals, which I mean, is obviously to be expected at some level, because it’s a you’re blocking mTOR. But what do you think is the pathway by which it decreases size? I know I noticed in the Gladius of paper, they talked about cell size is reduced in a dose dependent but are you also decreasing IGF1 and growth hormone. And all of that is that down the downstream pathway.
Speaker 1 14:23
I mean, presumably, right you you are sending a signal for that developing animal that creates sources are out of premia develop, right? And that growth hormone is and and downstream of that IGF, one are the signals to the body to grow and divide. And so, I mean, just my speculation is that you’re they are probably inducing something like the Snell dwarf, the infrared mouse model, where they have limited growth hormone at that at that time. Now, you know the way to really test that, obviously, I think would be to give them growth hormone while you’re giving them rapamycin to see if that, if you reduce that. An easy test to do that, but, you know, that’s what they did in Snell and Ames works to show that that was really what, why they live longer limiting at that point. I don’t know that. I think a lot of the stuff is speculation that based on those particular effects early on in life, right? I mean, as you said, they got it early on. They never got it the rest of time. If you’ve set something like growth hormone differently early on, yeah, it’s gonna affect lifespan long time. Raises novel ideas on how import may regulate early development with growth hormone IGF, one I don’t know about its translational impact, obviously something we want to avoid in humans. Yeah,
Speaker 2 15:40
I think in your 2015 paper, you started off doing one milligram per kilogram dosing. That’s a pretty aggressive dose. What was their rationale behind that?
Speaker 1 15:52
So part of it was based on the ITPs first 14 parts per million is in the ballpark of 2,000,014 Yeah. That’s what they originally recorded, right?
Speaker 2 16:05
I’ve looked at different translational animal to to primate or other, you know, mice to other animal studies. And I wasn’t sure on the calculations there could be, you’re targeting 40, 42ppm, or whatever it was, or 14ppm.
Speaker 1 16:20
So that was one part we were trying to talk target that. And I think following up from that, both from the ITP, from Matt’s lab, it’s shown that they can, mice can tolerate much higher doses and potentially added benefit. The other thing that we drove that on is we did an early pharmacology study pharmacokinetics, and we wanted to go lower doses for, I think, good reasons, right? And then with the side effects, we don’t want to get translational potential benefit, people say, a big, huge, high dose. But what we found is that much lower than one made per kilogram, the trough levels of grapple were ordering on undetectable. 24 hours after we gave them that dose, they were already out of that clinical even
Speaker 2 17:01
Don’t Marmosets have basically the same pharmacokinetics as humans, in terms of half life or not,
Speaker 1 17:10
you would think, but no, it’s, it’s probably more on par with the mouth estimated half life because we didn’t do full but it’s probably more on par with now,
Speaker 2 17:21
which was like 15 hours, I think
Speaker 1 17:24
probably around 24 okay, but it’s definitely not 48, 50 hours, you know what’s been reported in people.
Speaker 2 17:32
The dosing for marmosets is not, I mean, would you translate that to something similar in humans, or given that the half life you do maybe, is that like half the dosing?
Speaker 1 17:45
I mean, I think a safe estimate would be half of them, and the way we administer it is once daily, kind of like if they were taking a pill, right, right? I was in the yogurt, yeah.
Speaker 2 17:56
So we commonly see, and I’ve not seen a research study recently on the cause of this diarrhea and other side effects at higher doses like that. Do you not see any? It does sound like you see much in the way of side effects in these animals?
Speaker 1 18:10
Not really. And so it’s complicated, because marmosets get diarrhea. Normally. I’ve never seen a mouse get diarrhea, so I don’t know pharmacists do in lab. But rapid doesn’t seem to accelerate that, if anything, it might make it better in our animals. Just sort of anecdotally, we, you know, the things like the mouth stores we don’t see, but probably because we’re giving the encapsulated, the inherent, coded wrap up,
Unknown Speaker 18:38
or this is E-Rapa?
Speaker 1 18:42
yes, same stuff with the ITP, we initially thought we were getting a slight increase in hyperglycemia, but I don’t think that that’s a real I think it’s a transient effect.
Speaker 2 18:54
So obviously, in the study, you didn’t see it long term, but I was wondering, do you see that? Is there a short term rise in either lipids or glycemic responsiveness?
Speaker 1 19:09
We don’t know for sure, because we kind of had the limit how much we can do them. You know, we have to anesthetize them. You do have to restrain so we don’t want to cause too much stress. Oh, yeah, don’t know. We probably should do a separate study to test the timing effect. We know it two weeks afterwards, there’s no real effect, and we know that a few months, I think two months, was the early stages, there’s no real effect.
Speaker 2 19:42
okay, you you saw a weight loss. It looked like from most of the period, but then it seemed to rebound at the end of the study. Any idea what’s going on there?
Speaker 1 19:51
Yeah, that was a pilot study with with a fewer number of mice than we have in the in the main lifespan study. Okay? So. Yes, we kind of saw a similar effect. And no, no, at no certain points, it might be significant, but there was no sort of real clearance. Then I think it’s hard to say, I can’t say it’s not Rapa related, but there are a lot of factors. When these guys are animals, they’re kind of responsive to changes in, you know, weather conditions outside, because we’re not completely related or separated from the outside, changes the stress of somebody new moving in as animal people. So I think there are a lot of external factors we don’t always consider.
Speaker 2 20:34
I was interested in the accelerometer measure, is it basically like a Fitbit, and it measures the amount of, is it a pretty good proxy for the amount of exercise that a monkey gets?
Speaker 1 20:49
Yeah, it’s probably better approximates how much they move. But we don’t have a physical distance, right? And they’re not in a huge cage, it’s, I mean, it’s, it’s roughly six feet tall, maybe two or three feet wide. So it’s kind of constant around I would say it’s more of an activity than it’s interesting, because
Speaker 2 21:08
we do see a lot of reports on both dog owners as well as people that more energy and and, you know, more activity. And so I would have expected it’s you actually have rapamycin as a lower level of activity than the controls, which I found surprising. Yeah, and it may just
Speaker 1 21:27
be because they’re a caged animal too, right? I mean, people or dogs are outside social element to it as well.
Speaker 2 21:34
Did you do biomarkers and epigenetic or omics based testing at all to track epigenetic changes. Are you working with Horvath at all, or anything like that?
Speaker 1 21:45
We do. We publish the paper 2008, in part was to develop a clock for Marmosets. They have their own interesting weirdness that he brought up for us too. I mean, I can share separately from the wrapper. But so we developed the clock. We also pulled blood from the same end, and the medium length answer, answer is, is that wrapa In the blood did not affect that cloth, but there were certainly epigenetic marker specific ones that were all we need to follow up on what they are, what that might mean, but, but as a general overall trend. It didn’t shift the clock rate. Steve had some ideas. He said he didn’t seem to think blood was the best tissue to be using for it, and he very well may be right. We haven’t followed up on some of the other tissues we’ve collected, though, that that’s going to be what people are using. Is this their marker for the cloth. They’re not going to do liver biopsies, right, right? So, you know, I think it speaks to we still aren’t clear on what the value of those clots is, biologically, physiologically, they might, they might be good biomarkers, but we know that rapid and these groups didn’t affect right?
Unknown Speaker 22:57
Are you tracking this over time in current studies?
Speaker 1 23:00
We’ve collected blood from them We haven’t followed up, so they will. Some of the animals were on rapamycin that study for upwards of four or five years. Okay, so presumably we would have expected that have significant effect at that point, right? As I said, maybe that it’s not a clock rate, but specific markers that are most important.
Speaker 2 23:22
you measure measure trough levels. You don’t work at measuring peak levels?
Speaker 1 23:30
it’s an estimated peak, it was about 30 nanograms per ml, whereas the trough level is around seven to 10, so maybe three fold higher at the peak,
Speaker 2 23:44
and that, what’s the timing on that? Approximately, any idea,
Unknown Speaker 23:47
four hours
Unknown Speaker 23:50
do you track sleep at all.
Speaker 1 23:51
We don’t, just anecdotally, They have a home in cage on the side, they sleep in. We can look at it with video, but we don’t have anything beyond simply how much time the
Speaker 2 24:09
is their the standard sleep cycles like humans, and I would assume they sleep at night. And the accelerometer would probably tell you something, right?
Speaker 1 24:19
We have that, and it’s on for think we were in 48 hours. So we should have that very, very, going to be a very, very rough.
Speaker 2 24:28
We see a lot of reports of people, and I’ve taken rapamycin in the afternoon or evening, and it blows your sleep out for the can be very disruptive. And I’m not sure what the what the cause exactly is,
Speaker 1 24:43
yeah, and sometimes with the, you know, with the commercial drugs, to the adjuvants that are in there, so you don’t know if it’s necessarily the drug itself or something you’re reacting to. And I’ve heard that with other drugs, that was kind of like anecdotal reports. It’s not. With mice, whether they’re sleep habits or things like that. I don’t recall. I’ve never seen any studies
Speaker 2 25:06
Does your group have any theories on why there’s a sex difference in the blood levels? Yours was the opposite of what they saw in the mice and the ITP studies, is that right?
Speaker 1 25:19
No idea. But one thing that’s that’s strange about marmosets is that they are primary. So inutero, the male and female siblings share some of those. So when you when you pull this blood, you’ll see male and you’ll see female and, okay, drive females to be lower milk little. But it might just be the sample size that we did at that point. I need to follow up with our larger sample size and see if that’s true. I know that’s what we reported, but that’s what we had.
Speaker 2 25:48
You mentioned you did not find any inflammation reduction. How you measure that via What? CRP?
Speaker 1 26:01
yeah. So, you know, one marker the problem, yeah, problem for anybody working with marmosets is that a lot of reagents that we use for mice and human so forth. Okay, you know, most people, when they’re doing inflammatory profile, will use things like milliflex that have a lot of different antibodies, all the ILS all the DNS, those work from home, and it’s not that they don’t have il six or il that antibody, doesn’t we, we. So what we have to do is RT, PCR, and it’s really tough on blood cells, variability we but to get those tissues, we’re waiting until they die. Oh, it’s a mixed bag. I can say that with CRP, we didn’t see but CRP is not the only way you
Speaker 2 26:45
don’t see any hyperglycemia, and that’s just seems amazing, given the dosing levels. I’m just really shocked.
Speaker 1 26:55
well you know, and listen, we thought about this for a while, and I think part of this, you know, of course, they’re the increased risk of type two diabetes in people. I’m not going to discount that, but in large part, those people are compromised, right? And they’re on not just rap I don’t know that there’s any solid evidence that relatively healthy person on rapa is at any increased risk of death than anybody else in mice. It’s really clear that we give them rapid they become HyperFlex, at least with like, something like a glucose tolerance. I don’t, I don’t know that. It’s clear that it’s a baseline effect
Speaker 2 27:32
long term, if they remove the Raphe mice, and it goes away, right? It’s kind of like caloric restriction, it seems
Speaker 1 27:37
like. And there is, there’s lots of differences between a mouse and a marmoset or a person, but one of the big differences in physiology is that a mouse liver, which produces a lot of the glucose that we see when they’re stimulated, is huge in comparison to body size compared to people or marmoset, right? I mean, liver can make up five, 10% of the body weight of a mouse, depending on this. And if human are, honestly, it’s like one that’s part of this is because they just have. Mice have a big reserve of being able to produce glucose under those almost universally. But we also see that in humans, we do, we do. But again, I think it’s those people generally are confident. If there’s Healthy People taking Rappa that are reporting this too, I haven’t seen, you know, systematic data showing it covid True. Well,
Speaker 2 28:29
we see in our group, we’ve got, you know, hundreds of people reporting. We’ve got like, 10% reporting increases in blood glucose responsiveness, or
Speaker 1 28:43
normal resting, passing level, mostly responsiveness,
Speaker 2 28:46
but some basal level, but the but that’s during consumption, obviously. And we have testing, I’m not sure if we’re actually we aren’t specific. People aren’t specific in terms of this, is this near the trough levels, or is this at the peak level? Okay, so that’s, I guess, another point, right? Yeah.
Speaker 1 29:05
And it may be how the data is reporting too, right? If we in animal studies, typically, we’re looking at the average right and right probably find 10% that are higher in one or another, right? You’re seeing the sort of the same thing looking at the data in
Speaker 2 29:19
the different Yeah. Okay, those are all true. Groups are studying the level of autophagy caused by rapamycin, but we really don’t have a dose response relationship understanding yet. Do we? No,
Speaker 1 29:33
I don’t think so. Maybe in cell, but in animals, nobody systematically dying, and we don’t know how much is a good amount of boosting of coffee, right?
Speaker 2 29:43
So it’s all lot of lot of unknowns in that area. Okay?
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