A transcription of the interview / discussion:
I used otter.ai for this transcription and have cleaned it up a bit but havenāt 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 donāt have a biology background is also important too, right? I mean, not only for their perspective on the things that weāre putting out, but coming up with new ideas, right? Why not this approach? And I see some of the questions here, you know, theyāre insightful, the ones we sent, theyāre very insightful. And I think we donāt always have in our minds that direct, translational sort of aspect as basic biologists. So itās great to take ideas around with other folks. Oh, great, super
Speaker 2 0:31
I appreciate that. So youāve 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, Iām pretty well versed in it, but weāre not really following up on, you know, this molecule does this, this does that. Itās really, what benefit can we have on health and aging? And youāve 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 itās a long project, right? You were talking about primaries. Weāre not talking about mice, and some of itās 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 itās not really clear, right? If you did venture to me, no oneās really said hereās a time dysregulated, or that with age, itās always dysregulated in every tissue, right? Thereās, thereās sort of this assumption from, you know, maybe from invertebrates, maybe from mice, and itās just like a progression, and itās sort of the same everywhere. But I donāt think itās 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 thatās thatās where the money is. Itās my my knowledge, thatās 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, itās always sort of a static look, right at this particular point, thatās what it looks like. Weāve got a little better way to modulate them and look at response and sort of kinetics of that response, but cells arenāt a great model of humans, and
Speaker 2 3:04
we also donāt have much visibility in terms of tissue specificity or anything like that, really, right?
Speaker 1 3:12
itās 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 weāve 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 thereās no question my mind. I donāt think anybodyās really tested it beyond maybe some mouse work where theyāve 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 donāt think itās clear, but I think weāre 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 thatās thing, I think, not just with rapamycin, but all of Aging studies, we tend to make assumption based on data thatās been published that but we donāt make comparisons, and itās hard because you have lots of groups and lots of resources required, but so a lot of the stuff, weāre still at the very early stages. I mean, to be fair, human drugs, we canāt 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 itās 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 Iām 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. Itās all one sort of extension. So Iām 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 Iām sorry, you get a big response, because this transient pathway get as a very blunted response when rapamycin, it doesnāt take very long mouse models for that to once you remove rapamycin, within weeks, theyāre back to a normal sort of response to that. Okay, permanent shift, it likely is, as long as the rapamycin is available, itās itās causing this, because itās binding here in the washing and once you reach the half life of the drug, I think essentially, itās 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 itās going to slowly return back, you know. So thatās the direct effects on Mtor. But I think the indirect effects, if it had on epigenetics, well, because it boosted autophagy. Now youāve cleared out bad proteins, those will certainly remain until all that drove it are back. Thatās 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 thereās good reason to believe that itās the indirect effects, the downstream effects, that weāre really concerned about, not the direct effects on mTOR at the time, but weāve really separated those out scientifically for no for sure. So
Speaker 2
mTOR inhibition doesnāt really have any physiological effect on the uptake of the nutrients in the body. So if youāre, if youāre in a mouse or a monkey, and you feed it protein while itās while itās 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 donāt know of anyone whoās 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 donāt know. You know, the same thing could be said. Youāve 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 hasnāt been a lot of research on that yet. It seems like
Speaker 1 9:16
no, I donāt 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 donāt know, that we donāt conservative with my answers, because thatās the kind of I think scientist I am, and thereās a lot of stuff we donāt 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 itās being overwhelmed by the positive effects in other areas?
Speaker 1 9:54
Yeah, I donāt know. Because in part of it, I donāt know that itās. Still hard for me to discern. What are positive effects on changes in the microbiome beyond, okay, itās associated with good health. Limited Data that changing the microbiome directly does these things. And I mean, I think itās a growing field, but thereās 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 itās a whole different world too. Weāre talking about a mouse microbiome versus application to human I mean, we eat different things. We donāt 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 whatās your opinion?
Speaker 1 10:59
I donāt know about better. I think, I think I think theyāre different, and weāre 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, weāre modeling humans, and theyāre 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 weāre looking at that arenāt specifically towards general health. You know, part of this is viewing it like a clinical trial. Weāre 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 itās 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, Iāll have to come back to the mouse, because we donāt know. My general feeling is itās 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 Iām not saying that, that weāve 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 thatās, thatās my feeling now, thereās, thereās the counter to that is that these mice may be living with different diseases, that because theyāre mice, they donāt die, right? Diabetes or heart disease, heart disease, and we donāt know that. Hopefully weāll know more with mom and study because they do get they do get cardiovascular
Speaker 2 13:44
I donāt 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 itās a youāre 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 youāre 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 youāre giving them rapamycin to see if that, if you reduce that. An easy test to do that, but, you know, thatās what they did in Snell and Ames works to show that that was really what, why they live longer limiting at that point. I donāt 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 youāve set something like growth hormone differently early on, yeah, itās gonna affect lifespan long time. Raises novel ideas on how import may regulate early development with growth hormone IGF, one I donāt 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. Thatās 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. Thatās what they originally recorded, right?
Speaker 2 16:05
Iāve looked at different translational animal to to primate or other, you know, mice to other animal studies. And I wasnāt sure on the calculations there could be, youāre 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 Mattās lab, itās 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 donāt 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
Donāt Marmosets have basically the same pharmacokinetics as humans, in terms of half life or not,
Speaker 1 17:10
you would think, but no, itās, itās probably more on par with the mouse estimated half life because we didnāt do full but itās probably more on par with mice,
Speaker 2 17:21
which was like 15 hours, I think
Speaker 1 17:24
probably around 24 okay, but itās definitely not 48, 50 hours, you know whatās 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 Iāve 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 itās complicated, because marmosets get diarrhea. Normally. Iāve never seen a mouse get diarrhea, so I donāt know pharmacists do in lab. But rapid doesnāt 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 donāt see, but probably because weāre 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 donāt think that thatās a real I think itās a transient effect.
Speaker 2 18:54
So obviously, in the study, you didnāt 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 donāt 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 donāt want to cause too much stress. Oh, yeah, donāt know. We probably should do a separate study to test the timing effect. We know it two weeks afterwards, thereās no real effect, and we know that a few months, I think two months, was the early stages, thereās 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 whatās 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 itās hard to say, I canāt say itās not Rapa related, but there are a lot of factors. When these guys are animals, theyāre kind of responsive to changes in, you know, weather conditions outside, because weāre 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 donāt 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, itās probably better approximates how much they move. But we donāt have a physical distance, right? And theyāre not in a huge cage, itās, I mean, itās, itās roughly six feet tall, maybe two or three feet wide. So itās kind of constant around I would say itās more of an activity than itās 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 itās 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 theyāre 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 didnāt shift the clock rate. Steve had some ideas. He said he didnāt seem to think blood was the best tissue to be using for it, and he very well may be right. We havenāt followed up on some of the other tissues weāve collected, though, that thatās going to be what people are using. Is this their marker for the cloth. Theyāre not going to do liver biopsies, right, right? So, you know, I think it speaks to we still arenāt 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 didnāt affect right?
Unknown Speaker 22:57
Are you tracking this over time in current studies?
Speaker 1 23:00
Weāve collected blood from them We havenāt 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 itās not a clock rate, but specific markers that are most important.
Speaker 2 23:22
you measure measure trough levels. You donāt work at measuring peak levels?
Speaker 1 23:30
itās 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, whatās 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 donāt, just anecdotally, They have a home in cage on the side, they sleep in. We can look at it with video, but we donāt 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 itās 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 Iāve taken rapamycin in the afternoon or evening, and it blows your sleep out for the can be very disruptive. And Iām 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 donāt know if itās necessarily the drug itself or something youāre reacting to. And Iāve heard that with other drugs, that was kind of like anecdotal reports. Itās not. With mice, whether theyāre sleep habits or things like that. I donāt recall. Iāve never seen any studies
Speaker 2 25:06
Does your group have any theories on why thereās 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 thatās thatās 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, youāll see male and youāll 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 thatās true. I know thatās what we reported, but thatās 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 theyāre 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 itās not that they donāt have il six or il that antibody, doesnāt we, we. So what we have to do is RT, PCR, and itās really tough on blood cells, variability we but to get those tissues, weāre waiting until they die. Oh, itās a mixed bag. I can say that with CRP, we didnāt see but CRP is not the only way you
Speaker 2 26:45
donāt see any hyperglycemia, and thatās just seems amazing, given the dosing levels. Iām 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, theyāre the increased risk of type two diabetes in people. Iām not going to discount that, but in large part, those people are compromised, right? And theyāre on not just rap I donāt know that thereās any solid evidence that relatively healthy person on rapa is at any increased risk of death than anybody else in mice. Itās really clear that we give them rapid they become HyperFlex, at least with like, something like a glucose tolerance. I donāt, I donāt know that. Itās clear that itās a baseline effect
Speaker 2 27:32
long term, if they remove the Raphe mice, and it goes away, right? Itās kind of like caloric restriction, it seems
Speaker 1 27:37
like. And there is, thereās 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 theyāre 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, itās like one thatās 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 itās those people generally are confident. If thereās Healthy People taking Rappa that are reporting this too, I havenāt seen, you know, systematic data showing it covid True. Well,
Speaker 2 28:29
we see in our group, weāve got, you know, hundreds of people reporting. Weāve 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 thatās during consumption, obviously. And we have testing, Iām not sure if weāre actually we arenāt specific. People arenāt specific in terms of this, is this near the trough levels, or is this at the peak level? Okay, so thatās, 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, weāre looking at the average right and right probably find 10% that are higher in one or another, right? Youāre 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 donāt have a dose response relationship understanding yet. Do we? No,
Speaker 1 29:33
I donāt think so. Maybe in cell, but in animals, nobody systematically dying, and we donāt know how much is a good amount of boosting of coffee, right?
Speaker 2 29:43
So itās all lot of lot of unknowns in that area. Okay?
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