#10 - Matt Kaeberlein, Ph.D.: rapamycin and dogs — man's best friends? — living longer, healthier lives and turning back the clock on aging, and age-related diseases

Foreign.

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Find more information on today's episode and other topics@peterattiamd.com. In this podcast, I'll be speaking with Matt Kaeberlin at the University of Washington.

I met Matt about a year and.

A half, maybe two years ago through David Sabatini, who some of you may be familiar with just based on all of his remarkable work around MTOR and rapamycin. Matt's recognized globally for his research in the biology of aging. He got his PhD from MIT in the lab of Lenny Guarante, who's produced a number of notable folks in this field. He went on to do his postdoc at the University of Washington, and after completing his postdoc he has remained there and continues to run a fantastic lab. In this episode we're going to talk about his experience as the director of the Dog Aging Project, which, as its name suggests, focuses on the animal model of dogs for its research. Now, of course, this is really interesting because while fruit flies and yeast and mice are interesting, dogs are obviously much closer to us. And of course these dogs, because they're pets, generally have something really unique to us that virtually no other research animal has, which is they share our environment.

And this puts them in a pretty.

Unique spot that even the rhesus monkeys studied in the NIA Wisconsin Project didn't have going for them.

Now, if you haven't already done so.

I'd recommend listening to the podcast that I did with David Sabatini because that will get you some of the background on MTOR and rapamycin. I think we get a little technical in this podcast, but I suspect it's something that if you've listened to that other one with Sabatini, you'll have the background to follow. And we talk about a bunch of other things that people seem to be interested in as well, beyond rapamycin and aging. We also talk about nad probably get into sirtuins a little bit.

The other thing I really enjoy about.

Speaking with Matt and I had so many discussions with Matt, you know, over meals and stuff Where I think to myself after, man, I wish that was recorded because one, I want to be able to hear it again. And it's the kind of stuff that people are always asking me. And I think Matt just has such an amazing way of thinking about this stuff. Matt's work is really remarkable because it's actually focusing on healthspan. It's easier in some ways to study healthspan because you can study it over a shorter period of time. And in particular, when you look at the cardiomyopathy model, meaning it's a type of heart failure that dogs experience, and you look at how rapamycin can improve ejection fraction, you can get these answers in a really quick period of time, in fact, much quicker than I think Matt even expected. And we talk, of course, about cancer, heart disease and Alzheimer's.

Dis.

If you're interested in rapamycin, if you're interested in mtor, if you're interested in longevity, I think you're going to find this interview very interesting. It was actually recorded initially in December of 2017 as part of the interview series I was doing for my book. And we may at some point throw up the video as well. You can find a lot more information in the show notes, of course. And without further delay, here's my conversation with the remarkable Matt Kaeberler.

Matt, thank you so much for being here. It's really fantastic to be able to talk with you in this format. I've been a huge fan of your work for many years now. Obviously I know quite a bit about your background, but I think it'd be great for the listeners to kind of get a sense of what did you study in college, how did you end up doing your PhD, where'd you do it, and most of all, why did.

It get you where you are?

Sure. So I got undergraduate degrees in biochemistry and mathematics at Western Washington University, and then I went to graduate school at mit, the biology department there. And my background up till that point had been in biophysical chemistry. So I really went to graduate school thinking I was going to work on structural biology or x ray crystallography or something like that. And I heard a seminar by Lenny Guarenti, who I ended up doing my PhD thesis with during my first semester at MIT, where he started talking about how his lab had begun working on the genetics of aging and trying to understand what are the factors that influence the rate of aging. And this was in yeast. That's what his lab worked on at that point. And it was really almost like an aha moment. Where it just clicked with me that it was really fascinating that you could use genetics and molecular biology and biochemistry to study something as complicated and fundamental as the biology of aging. And so I got fascinated by the topic. I ended up going and talking to Lenny and then subsequently doing my thesis research with him. So it was really that moment of hearing him talk my first year in graduate school that got me interested in aging. And that's what I've been fascinated by and passionate about since then. And so then I went on and did a postdoc with Stan Fields at the University of Washington, also working on aging, and then ultimately started my own lab. So it's been almost two decades now that I've been working on this problem.

And if my memory serves me correctly, when you were in Lenny's lab, who's obviously no stranger to probably people who are listening to this, you worked on sirtuins, correct?

Yeah. So my thesis work was actually the work that first showed that if you activated or overexpressed a sirtuin, in this case it was sirtu, the founding member of the sirtuins in yeast, that you could extend lifespan and slow aging. And obviously we were very excited about that, especially when a postdoc who was in the lab at that time, Heidi Tissenbaum, about a year later, showed that you could also overexpress the worm version of Sirtu and extend lifespan and C. Elegans. And so that was really the first example of a conserved genetic modifier of lifespan across two widely evolutionarily divergent organisms.

Because it was a relatively similar version of Sirtu. So more different than the DAF stuff that we saw in C. Elego versus the IGF that we would see in some of the higher mammals.

Right. So at that time, we knew that DAF2 could extend lifespan. And C. Elegans, I don't think we.

Knew it was working on a similar mechanism.

That's right, yeah.

Now, I think one of the things that makes your work so interesting is that you work on dogs.

Right.

How did you end up doing that?

Yeah. So that's relatively new in my career. It was about four years ago when Daniel Promisloe moved from the University of Georgia to the University of Washington. And Daniel had started thinking about dogs as a model to understand how genetic and environmental factors influence the aging process. And I'm a dog person. I've always had dogs. I have three dogs now.

That qualifies.

Yeah, right. And I had never really made the connection between dogs as companion animals and dogs as a model for understanding aging until I talked with Daniel about this, and through those conversations, it occurred to me that not only could we understand genetic and environmental modifiers of aging, but our pets could actually serve as a transformative next step in testing some of these things that we know work in laboratory mammals, like mice, but have never yet been taken outside of the laboratory. And so it was really that sort of connection in my mind about three years ago that propelled me to really move forward with the dog aging project, one major goal of which is to actually do this, to take some of the interventions that we know extend lifespan and slow aging in the laboratory and test whether they have that effect in the real world in a larger mammal that's also very socially relevant. I think that that's an important aspect of this. People love their dogs. If we can actually slow aging in people's pets, that's going to have a huge impact both on the quality of life for the pets and the owners, but also the way that people think about the biology of aging. They're going to believe it a lot more if they see that their dog is living longer and aging more slowly than just reading an article in the New York Times or wherever.

Right, right. And it's, you know, I sort of. When I talk about this, I generally talk about these four classes of eukaryotes, going from, you know, yeast to flies, worms, and mammals. But usually when we talk about mammals, we're talking about mice. Absolutely. They have a couple of problems, at least.

Right.

One problem is they're generally. Especially if they're inbred. I mean, they're homozygous at all low size. So you have to question their applicability to us in terms of their susceptibility to disease. But then the second point, which you alluded to, which I think the dogs might be the first laboratory study that gets out of this, is they don't live in our environment. Right. The mice, that is. So the dog truly lives in the environment you and I live in, which, at least in theory, should be the environment we care most about preventing aging in.

That's right. I think that environmental variation is hugely important. The genetics is also important, and dogs are. So there's a couple things that are worth mentioning. Dogs have some of that same genetic homogeneity if you look within individual purebred breeds. But we also have many purebred breeds that are widely divergent, both genetically and morphologically. All you have to do is look at a Chihuahua and a Great Dane. Right. To see that divergence. But we also have this really Interesting and complex mixed breed population that's a combination of all these different genetic variants that each breed has. So we have kind of the best of both worlds in the sense that if you want to do a study in a relatively genetically restricted background, you can do it in a specific breed or set of breeds. But. But if you want to capture that genetic variation, all you have to do is look in the mixed breed dogs. But the environmental part of this, I think is probably the most important from a what are we going to learn from dogs that we can't learn from mice perspective. As you said, dogs really share our environment to a greater extent than any other animal, maybe with the exception of cats. And so they're drinking the same water, in fact, may not even be drinking the same water. Most people may be drinking worse water. Yeah. Right. Are not going to give their dog bottled water. Right. But they're breathing the same air. If you smoke, they're experiencing that, or someone in the house smokes, they're experiencing that secondhand smoke. So they really do capture most of our environment. The diet is about the only place where dogs don't quite have the same sort of nutritional diversity that people do. But with the exception of diet, the environment is pretty close and we have.

A pretty good idea of how we die. I could actuarially map out how you and I are going to die based on our age and a whole bunch of other factors. Again, it's probably an oversimplification because of this species diversification, but as a general rule, how do dogs die?

Yeah. So I'll make one quick comment on that. First of all, that I think you're right, that we do know what diseases most often kill people. And that's important information. I'm not sure it's the most important information because what people die from does not always equate to what they die with. Especially today, most people are dying with.

With chronic diseases.

Yeah. Multiple comorbidities. Right.

Y.

Just because you may die from heart disease, it doesn't mean that you didn't have kidney disease or something moving towards kidney disease or diabetes. So I think it's important to appreciate that you can have multiple diseases and only one of them is probably going to kill you. Having said that, it is useful to think about do dogs die from and with the same age related diseases that people do. And the answer is in general, the equivalency is pretty good. So dogs get all of the same age related diseases that people do. They don't get them at the same frequency necessarily. So one big difference is in dogs there's actually relatively little vascular disease, which is a major killer in people.

So specifically atherosclerosis, whether it be peripherally or cardiovascularly, doesn't seem to be as prevalent.

Does not seem to be as prevalent. But there are breeds that die from. From heart disease and certain forms of heart disease. Cancer is probably the most common cause of death in dogs, and big dogs tend to get more cancers than small dogs. But across all dogs, cancer is probably the number one cause of death. Kidney disease is a major cause of death in dogs.

Is the etiology of that kidney disease related to blood pressure? Is it related to some other nephrotic syndrome that's otherwise unidentified?

I don't know the answer to that. Yeah, I don't have the veterinary background to answer that. One of the things that's interesting in dogs is that there seems to be a little bit of a debate whether dogs really get Alzheimer's disease. They clearly get dementia. Some dogs do, and they clearly show cognitive decline. It's not completely clear whether they get what would be clinically diagnosed as Alzheimer's disease in people. Apparently they do accumulate a beta in the brain. Whether they get the plaques and tangles still seems to be a little bit up in the air. And there are people studying that. But at least at the level of cognitive dysfunction and dementia, it's clear that dogs experience that with age as well. So for the most part, they do develop the same age related diseases. The increase in risk for those diseases goes up essentially exponentially, just like in people. But the relative prevalence of specific diseases is not always the same and can also be breed dependent. So there it is certainly the case that within purebred dogs, different breeds, different have different predispositions to certain diseases, which is exactly what you'd expect based on the genetics.

So basically, they're probably getting a little more cancer, significantly more renal failure as a proximate cause of death, as opposed to dying with renal insufficiency, which to your point, I think a lot of humans do. They're getting a lot of heart disease, but it sounds like it's more cardiomyopathy and or valvular disease, but not atherosclerotic or ischemic disease. And they unfortunately die of accidents just as humans do, which would still probably be in the top four for humans and dogs, I'm guessing.

Yeah, and actually that's an interesting point. You're right. What I haven't seen great data on is the age distribution of death due to trauma. I suspect it's going to be mostly younger dogs, but I don't know for sure. So that's actually an interesting question. It is absolutely true. The other thing that is worth noting about dogs is it's actually the case that most pet dogs don't die from an age related disease, they die from euthanasia, which is a difference between dogs and cats.

Yes, that's a great point. Well, let's turn the discussion now to what you do about this. I've been a big fan of rapamycin and Tor and that entire pathway for several years now. It's become almost an obsession, if one can have an obsession that's not pathologic. And I think what attracted me to your work a couple of years ago, probably David Sabatini pointed me in your direction. So maybe three years ago, it was actually after the manic paper came out in, which was around 2014.

Right. Which was sort of the first really.

Interesting look at the human data. I want to come back to that paper as it relates to immune function and stuff, but maybe give us a sense of how you decided that the next logical step was to actually test the God molecule, as I like to call it, Rapamycin in this context in dogs.

Yeah, yeah. So again, this was all happening about three years ago when I first sort of made the leap to thinking that we could test interventions in dogs. And actually that's not, at least for me, was not an immediate. It wasn't immediately obvious to me that we should. So you really have to think as soon as you start talking about bringing trial of a drug out of the lab and into the real world, whether it's the human clinic or the veterinary clinic, in the context of aging, you really have to start to think about safety and side effects. Right. Because there is a very low tolerance for side effects when you're talking about treating a healthy person or dog. So that was the first thing that I had to really come to grips with is could we do this safely?

And especially when we were clear you were thinking we're not going to take dogs that are already sick.

That's right.

We were going to take healthy dogs.

That's right. I mean, for me, as somebody who is fundamentally interested in the biology of aging, the intent is to slow aging in people before they get sick. Right. To keep them healthy longer. So in many ways it's the opposite of traditional medical approach biomedical research where normally historically, we wait until people are sick and then we try to cure their disease. This is the reverse of that. Right. So that's right. So because we are talking about intervening in a healthy person or a dog, the tolerance for side effects from a regulatory perspective or even just public perception is very low when you're talking about a healthy person. Now, you know, the way I view that, first of all, I think that is we need as a society and within the scientific community to have a discussion about this, because I think that we need to recognize that a healthy 70 year old is not the same as a healthy 30 year old. Right. And we know what's going to happen if we don't do anything about aging. So my personal view is that there should be a tolerance for some level of risk if the outcome is going to be 10%, 20%, 30% more time spent in good health. That's a discussion that we haven't had either at the regulatory level or at the society wide level. But I think we need to have. So I digress a little bit, but I had to go.

I think that's one of the most important points you could make. And I sort of think a lot about this because my colleague and I were discussing this the other day, which was, you know, you take this oath at the end of medical school, the Hippocratic oath, the first thing you learn to say is first do no harm. And I think the spirit of that is excellent. But I also think it's highly impractical in a world where it forces you into binary thinking, which is we will only undertake interventions that are guaranteed to have no harm and otherwise we will do nothing, regardless of the outcome. Which of course is not practical. We live in a probabilistic world where the probability of harm is not 0 or 1, but rather it's a continuum between 0 and 1. And you really need to take a risk adjusted approach.

That's right.

Two outcomes. So I mean, I think that's an accurate point.

Yeah, that's exactly right. So I had to kind of go through that thought process in my own head though, and especially thinking about moving into pet dogs where because of the way that many people feel about their pet dogs, you have to be as sure as you can that you're not gonna hurt anybody's pet in the first place.

They like them more than their friends.

Yeah, absolutely right. Yeah. People love their dogs. Right. A lot of people feel similarly about their dogs as they do about their kids. Right. So you kind of think that's kind of the way I thought about it is could we do this in somebody's child? And so with rapamycin, there is a perception out there that I don't share. But there's a perception out there that rapamycin has lots of side effects based mostly on the human clinical literature, which.

To be clear, is generally in transplant patients.

Transplant patients taking high doses and taking lots of other drugs. Yeah. So sick people taking a high dose of rapamycin in combination with other medications. Right. And it does have side effects. There's no question about that. But one of the things that's come out of my own research and other research in the field is that both the benefits and the side effects are strongly linked to dose. So one question was, is there a dose of rapamycin that will have beneficial effects in the context of healthy aging without significant side effects? That was an unknown. I think that we're getting to the point where we're pretty sure that that's the case. At least you can get some of the benefits of rapamycin and we can talk more about the data that support that. But at that point, it really wasn't. It really wasn't clear.

And when you say the dose, do you think that the peak or the trough play a bigger role in toxicity?

Yeah. So that's still an unknown, but I think the data that are out there suggest that. That the trough levels are most strongly correlated with side effects. Now, what is most strongly related to health span or lifespan? There's no data, as far as I know. And this is an area where I think there's a lot of work that could be done and should be done, exploring more broadly, even in laboratory animals, in mice, the sort of dose response and dose timing space for where do you get the biggest effects on lifespan or specific measures of health? Where don't you get any effects? Can you uncouple, say, the improvements in heart function from the improvements in immune function by changing the dose or the timing? There really has been very little done on that.

Now, maybe for the listener who's not familiar with the pharmacokinetic discussion, let's explain maybe trough and peak. How do we think about those dose?

Yeah. So when you give a medication, say a rapamycin pill, when a person takes a rapamycin pill, there will be a rapid increase in the levels of the drug in the blood. And if they don't take another dose, then the drug will start to get cleared and it will go down. And so if you're taking a pill every day, as soon as you take the pill, the blood levels go up and then it starts to get cleared, and then the next day you take the pill. It goes up, and then it starts to get cleared. So if you were to take the pill every other day, it would go down further before you get that spike again. So the spike, the top of the spike is the peak level, the bottom before you take the next dose as the trough. And so the little bit of data that's available, and again, there's not much data for different doses of rapamycin in people who are not also taking other drugs. So the combination here is both dose and rapamycin as a monotherapy. And so really, the only study that I know of that looked at this really at all actually didn't even use rapamycin. They used a derivative of rapamycin called RAD001 or everolimus. That's the manic study that we talked about. So there's really no good data in people on different doses of rapamycin as a monotherapy in healthy people. So we're kind of stuck looking at the data that we have. So in Joan's study with everolimus or RAD001, they gave the medication to healthy elderly people, and they tested three dose and delivery combinations. So one of them was, I believe, 20 milligrams once a week.

Once a week.

One of them was 5 milligrams once a week, and one of them was 1 milligram a day. Right. And this was looking in the context of immune function as measured by a flu vaccine response. So the outcomes were that, I think at all three doses, they saw evidence for improved vaccine response, which was consistent with prior data in mice that immune function is improved by rapamycin.

Pause for a moment. That's still a bit counterintuitive to the layperson. When I say the layperson, I mean like the layperson who still thinks about.

Rapamycin or actually, lots of physicians.

Sure. Because we think of this as an immune suppressant. And yet they took this drug in monotherapy under a different dosing schedule than a transplant patient would take it. And we saw an improvement in their T cell function. The same cells that we tend to knock out in a transplant patient.

Yeah. So let's come back to that, because that, I think, is. That's an important issue, but it's complicated just to come back to the trough levels and side effects. So they saw evidence for efficacy with every delivery method, although they got, I think, the best efficacy at the five minutes a week, five once a week. But they also had the lowest side.

Effects, and the most side effects were the ones every day or the 20s, once a week.

I think it was at the 20s, once a week, but I can't remember for sure. It was pretty comparable. So the first thing to say is none of the side effects were bad by clinical standards. None of the people dropped out of the study because they were taking the drug, which is, I think, a pretty good indication for how tolerable the drug is. So even though they did detect some side effects, they really were not serious. They weren't even serious enough that they were uncomfortable and people stopped taking the drug. So I think that that's really the only evidence that we have that I know of, that it's really the trough levels that drive side effects. So I kind of think that's probably true, but I'm not confident and I really think we need more data to know for sure.

It's certainly true in other classes of drugs. When you look at gentamicin, for example, and the ototoxicity or nephrotoxicity, it's a trough problem, not a peak problem. That's why you have to make sure the patients clear it before you redose it.

So there are other reasons to believe that as well. Yeah. So now coming back to this immune function, there was a washout, right? There was. So there's a couple things again there to consider. So the data suggesting that rapamycin can act as an immunosuppressant, again, is almost exclusively based on very, well, I shouldn't say very high doses, higher doses than were tested in the Novartis study in people who are also taking other drugs that probably are true immunosuppressants, typically at.

Least two, if not three other drugs in the cocktail.

So it's really not clear to what extent rapamycin as a monotherapy in healthy people has immunosuppressant properties. And the data in mice, I would say, is mixed. It really seems to be the case that for some forms of immune challenge, at high doses, rapamycin can enhance susceptibility to infection. For other forms of immune challenge, it enhances function. But again, those studies are almost always done at very high doses of the drug that are even much higher than you would give to a person. So it's an unknown. Now, what seems to be the case both in mice and people is that short term treatment with rapamycin in an old mouse or an old person followed by a two week washout where they stop taking the drug. When you then test immune function, at least as measured by a vaccine response, you get a better response. So one model would be that the Treatment with rapamycin is restoring immune function in an aged animal, probably through enhanced stem cell function, although I think that that hasn't really been demonstrated clearly. And you might need that washout period. If there is an immunosuppressive effect, you might need that washout period to be able to see that rejuvenation and immune function. Again. That's really speculative though because nobody's done.

In Joan's paper did they do. I'm sure they didn't actually, now that I think about it, they didn't have enough people in the study. Would have been very interesting to have seen the immune challenge without the washout.

Yeah, they didn't subset now. So I was just going to say nobody's done it in either mice or in people. That actually would be a fairly easy experiment to do in mice. The problem is you would never get an NIH study section to fund that experiment because we already kind of know the answer that rapamycin works. They wouldn't be viewed as an. Even though it's really important from a translational perspective, it wouldn't be viewed as innovative or important enough for somebody to fund a grant to do it. So it's an unknown. And I don't know how long it'll be till we actually get the answer to that question.

Now bringing it back to the dogs. One of the challenges of course in leaping from mice to dogs is mortality becomes difficult to study. You have an animal that lives a lot longer, whereas mice, you know, you can get a longevity answer in months if you select them correctly in dogs, if, if you want to study them for true hard outcome of death, it's something they want to study. So you pick a proxy.

Well, you can.

Yeah, I'm saying the shortest path would be, let's look at organ function or something else.

Right. So what we did, so we've done one study where it was a 10 week study of rapamycin in middle aged healthy companion dogs. And we chose heart function as our short term measure. And that again was based on mouse data where two different, actually three different labs now have shown that if you take a 20 to 24 month old mouse, that's maybe the, that's like a 40 year old. No, it's more like a 60 to 65 year old person that if you just look at the heart of a 24 month old mouse compared to, let's say a six month old mouse, you can see declines in heart function just like you can in people. And the parameters that have been studied with respect to rapamycin specifically are mostly measures of left ventricular function. So ejection fraction, fractional shortening, things like that. And this is done by echocardiography, so it's relatively non invasive. So you can see a decline in function with age. And what has been seen now in three independent studies is that six to ten weeks of treatment with rapamycin is enough to cause those measures of heart function in the old mouse to go back about halfway to what you would see in a young mouse. So it doesn't bring you all the way back to a teenager, but it gets you back to, you know, maybe a 35 year old heart if you're doing this sort of mouse to human equivalency.

And just to be clear, that was how many weeks?

So the studies have varied. It's between six and 10 weeks. All of them saw improvements. It's not clear whether you get bigger improvements by longer treatments. So it's kind of in that range.

And did these animals also require a washout to see the benefit?

No, no washout. So these are measures of heart function taken while the mice are still on the rapamycin.

And these animals were dosed daily.

So yes, all three of those studies used the encapsulated rapamycin in the diet. So they were getting the drug daily and it was not. And this is where it gets a little bit complicated because I'd say 85% of the studies on aging or age related functional measures in mice with rapamycin have been done with an encapsulated form of rapamycin in the diet. We call it Erapa. And so that's different from a pill. Right, because mice are going to eat throughout the night, so they're probably not experiencing exactly the same pharmacokinetics that you would experience from a pill. Or the other kinds of experiments that people have done in mice have been injections where you get this rapid peak in the drug and then a pretty steep drop off as the drug starts to get cleared. Nobody's ever done the 24 hour measures of rapamycin blood level on the mice getting era. So I don't know how different it is, but it's probably different in the sense that it's probably a more stable level of rapamycin in the blood for a longer period of time.

I mean, I would expect that the ERAPA animals are going to have lower peaks and higher troughs. I mean, that would be.

That would be the expectation. Yes. But I don't know that anybody's ever really carefully looked at that. So, having said that, I think all three of the studies that looked at heart function in mice used the Erapa, and it was a pretty low dose of the Era.

How many milligrams per kilogram?

It's 14 parts per million in the food. I don't remember off the top of my head what that works out to in milligrams per kilogram. I feel like it's in about the 2 range.

Wow. That much?

Well, yeah. But again, you have to keep in mind that you can't translate the dosing across species very well. 2 milligrams per kilogram in a mouse is not going to be the same as 2mg per kg in a person or it doesn't happen.

Because the dogs and the humans, I suspect, are probably a lot closer than the.

You would think so, although I don't know that there's actually good evidence to support that. So certainly in terms of body size, if you're talking about a bigger dog, they're closer in terms of metabolism of the drug, though, that I imagine could vary.

It's hepatically clear.

I don't know. That's a good question.

I'm pretty sure it's not renal P4.5. Yeah, it's going to be in the liver. So how did you ultimately come up with both a dose and a schedule of delivery for your first trial?

So this again goes back to this question that I was asking myself about can we do this safely? And there's very little data in dogs on rapamycin at all. And just like in people, there's almost none on rapamycin as a monotherapy in healthy animals. So I started basically digging and talking to as many veterinarians as I could to find out what was known. And I was very fortunate to actually be able to get in touch with a veterinary group at the University of Tennessee who was studying rapamycin in dogs who had had hemangiosarcoma of the spleen. And so they had done the pharmacokinetics and had developed a dosing strategy that they feel is extending life expectancy in dogs who have had hemangiosarcoma of the spleen and where they weren't seeing side effects and they had had some of their dogs on rapamycin for more than a year. So we were pretty confident, or I was pretty confident after talking to them that if we took their dosing strategy that we're unlikely to see any significant side effects over a 10 week period. And so I said that cardiac Function was the functional endpoint that we were using. Really the goal of that 10 week study though, was to confir, this is a phase one, basically, that we could do this, that we could get people to participate, they would actually give their dog the medication and that we didn't see any significant side effects. So I was pretty confident that based on their dosing and delivery protocol, we at least wouldn't see any severe side effects in a 10 week period.

Did NIH fund that study?

Well, not directly. So we got a little bit of NIH money because we have one of these Nathan Schoch Centers of Excellence in the Biology of Aging. And we had a small amount, it was on the order of a few tens of thousands of dollars left over from the prior year. And so I asked Felipe Sierra, who's the head of the Division of Aging Biology at nih, if we could use that surplus specifically for this project. And he was kind enough to agree to that. But it wasn't. We didn't write a grant and get.

A grant, but you sort of bootstrapped this thing.

Yeah. So actually I paid for this study mostly out of funds that I had gotten because I was recruited to get a job somewhere else. And as part of my retention package, the University of Washington gave me some money that I could spend on whatever I wanted to. And I thought this was a good way to spend the money.

So what did that 10 week study show?

Right. So first of all, I'll go back to the dosing before I tell you. That's right. Yeah, yeah. So I talked to this group at the University of Tennessee. And so their strategy was 0.1 milligrams per kilogram given three times a week, Monday, Wednesday, Friday. And that made sense to me in light of what we were just talking about from the manic paper where it seems like if you give an extra day to let the trough levels get down, it made intuitive sense.

Yeah. You can reduce the toxicity.

Yeah. So I don't know that that's the case, but it made sense. And so that was the dosing strategy that we went with for our highest dose. And then we also tested a dose that was half of that, so 0.05 milligrams per kilogram again, Monday, Wednesday, Friday. So we enrolled dogs into this study, this was a very small study, all at a private veterinary specialty clinic in the Seattle area. And so the dogs had to be at least 40 pounds and at least six years old, and they could not have any pre existing conditions. So again, this is a study of Healthy aging. We wanted healthy dogs coming into the study, six years old, because at that weight range, we figured that that would be roughly the human equivalent of 55 years. Maybe big dogs age faster than small dogs. So that's why we had the weight criteria. So we wanted to have a population where we expected there would be the potential for some age related functional decline, but that we wouldn't get a high proportion of dogs.

Yeah, you don't want to be too close to the edge of the cliff.

That's right. That were really sick. So one thing we found, though, that was unexpected was that about 20% of dogs in that age and weight range actually have asymptomatic heart disease that you will see if you give them an echocardiogram, but you won't detect from a stethoscope exam. And that was. In hindsight, it kind of makes sense. Right. Again, heart function is going to go down with age. And where you decline, like what is the clinical threshold that we call disease is sort of a moving target sometimes. Right. If a vet with a stethoscope takes a heart murmur and then they give the dog an echocardiogram and they see regurgitation, they'll call that heart disease. If they don't hear a heart murmur, nobody's gonna give their dog an echocardiogram. Right. It's expensive.

Yeah. And I'm not a cardiologist, but I can't imagine the day would ever come where using a stethoscope I could detect, you know, a low ef, like to the tone of. Yeah. I mean, unless there's.

You would not get that. No, no. It's really the regurgitation that they're hearing as a heart murmur. And that was most of the dogs that we ended up having to exclude. We had to exclude because they had a preexisting valvular regurgitation that came out from the echocardiogram. But this was actually a discussion that we had to have with the cardiologist. The cardiologist initially went into the study with the feeling that if there's any regurgitation that's beyond trivial, that that's heart disease. And so when we started to see dog after dog show up with this level of regurgitation, we had to have this discussion, what's normal aging versus disease. Right. And so just because of the way that the protocol was written, we ended up having to exclude about 20% of dogs because they had underlying heart disease. So we had 40 dogs come into the study for their first exam. That was Our target number, we ended up having 24 go all the way through the study.

Most of them three groups, two doses plus a placebo.

Yeah. And they ended up not being evenly distributed. And in part this was because. In part it was because it was my first clinical trial that I'd ever done and I didn't plan for the fact that we would have to exclude as many dogs as we did. So the way it ended up was we had 8 placebo, 11 high dose and 5 medium lower dose. Yeah, right. That went all the way through. We only had one dog leave the study and that was because the owner just stopped giving their dog the medication. And we had one dog where the owner gave their dog the wrong amount of the medication, which, ironically enough, physician low. So the dog was randomized into the high rapamycin group and ended up getting one quarter of the expected dose. So other than that, there was fantastic compliance. All of the owners did what they were supposed to, came in for their exams. So the main outcomes of this study were one, there was no evidence for increased side effects. So the owners filled out weekly surveys that there was a long list of.

The dogs didn't fill them out.

Well, you never know. There was a long list of did your dog experience any of these things? And then there were a couple of just open ended questions, you know, do you feel like you observed any positive changes or negative changes in health?

Did the group in Tennessee see the aphthos ulcers in their dogs? Because that seems to be. I remember as a resident when we would give rapamycin to the kidney transplant patients. I mean, the biggest complaint by far was those aphthous ulcers.

Yeah. And we didn't. Again, in hindsight, we probably should have done a better job of looking, but we did not have any evidence that that was happening. The only thing that initially I thought that maybe we were detecting that was because we had several owners. And this was while the study was still blinded, so I didn't know which dogs were which at this point. We had several owners report that their dog was drinking a lot more water and a couple of them actually came in for urinalysis and stuff like that. And so I thought maybe that could be like a dog's response. If they have sores on the inside of their mouth and they're uncomfortable, maybe that would be the sort of the canine equivalent of how they would respond to that. As it turns out though, when we unblinded the study that reported observation of increased water consumption was equally spread between the placebo and rapamycin groups. So I don't know if that's happening in dogs. That's something we'll look at in the next phase. So, again, for all of the side effects though, that we surveyed owners on no difference between treated and untreated, the blood chemistry showed no significant changes with rapamycin, which was actually a little bit surprising.

Did you do a glucose tolerance test?

We did not do a glucose tolerance test, in part because we wanted to keep the, the number of assays that we were asking the owners to subject their dogs to as small as possible, make it as non invasive as possible for the animals. We did get blood chemistry at week, you know, before randomization, at week three and at week 11. So within one week of coming out of the study and we saw improvements in heart function. Now, I will say it's a small cohort. They were on sort of a borderline of statistical significance. So two of the three measures that we had as our primary endpoints, the three measures were ejection fraction, fractional shortening and E to A ratio. And again, that was just coming directly from the mouse studies. Two of those three were statistically significant. One was P value of 0.06.

Yeah, but I mean, you must have been underpowered on anything.

Absolutely, yeah.

No, it's amazing you saw significance.

I agree. Yeah. So I think the way I view this is it's about the most positive outcome that we could have hoped for, clearly needs to be replicated, but at least the changes are going in the right direction. And a couple of interesting things. When you actually look at the heart data, it very much looks like the dogs on rapamycin that got the biggest benefits were the ones that started with the lowest function, which is not surprising. But that also is encouraging because that's kind of what you'd expect. Right. The dogs that have undergone a greater age related decline are likely to be the ones that are going to get the biggest benefit from a treatment that's actually affecting that. So that was really encouraging. And then we actually had one Doberman Pinscher in the study. And this turned out to be interesting because Doberman Pinschers as a breed are highly prone to dilated cardiomyopathy. Something like 60, 65% of Doberman pinschers will develop dilated cardiomyopathy as they get older. I wasn't really aware of this literature going into the study, but it turns out that many Doberman Pinscher owners will actually give their dogs echocardiograms or electrocardiograms routinely as they're getting older to try to detect dilated cardiomyopathy as early as possible. And the owner of this dog didn't tell us this before she came into the study, had actually been aware of this and was giving her dog echocardiograms before coming into the study. That dog had low cardiac function, but it was not yet to the point where it was clinically.

Clinically detected. Yeah.

As dilated cardiomyopathy. So our cardiologist in the study also not knowing the dog's history of having prior echoes, did not flag the dog as needing to be excluded. So the dog was randomized, it just happened to be randomized into the higher rapamycin dose. And its function was. That was one of the dogs where we saw the largest improvement in function. What is interesting about that is the owner then, after the study was over, continued to get echocardiograms every three months and has shared that information with us. And so it's really, it's an n of 1, but it's a really fascinating sort of case study because you can see the dog's cardiac function declining. Then the dog comes into the study, gets rapamycin, it shoots up, it's quite a dramatic increase. And then within about.

What was the increase in ef, do you remember?

I. I don't remember. I don't want to say the exact number because I don't remember off the top of my head.

It sounds like it was a big deal.

It was from the borderline of being occult dilated cardiomyopathy up at least 10, 10%.

10%? 10 absolute percent.

That's right.

That's enormous.

Yeah. Well, back into the normal range. Yeah.

And has that patient, or that patient, has that woman shared with you what the resulting decline in EF has been since the trial? So about.

I've got the data out to about six months and we're just now trying to reconnect with her to see if, if she has additional data that she'll share with us. By about six months out, the ejection fraction and fractional shortening were back right at the point when the dog came into the study. And at that point her cardiologist diagnosed the dog as occult dcm. So clearly going down the path to dilated cardiomyopathy.

So the million dollar question in a study like that or in a case like that is if you had to guess what would be the ideal way to take care of that dog to delay the onset of cardiomyopathy as long as possible, would it be just keep this dog on that dose three times a week in perpetuity, would it be give the dog a 10 week holiday? 10 on, 10 off. 10 on, 10 off.

Right.

So it's clearly 10 on every six months is just a seesaw factor.

If everything's working the way that we think it is. Yeah. My guess is that the default there would be to keep the dog on the drug unless you start to see side effects. Right. So continue to monitor by echoes every three months. And unless you see side effects or unless you see something else that makes you worried that rapamycin is having a negative effect, just keep the dog on the drug. But it's an unknown as to whether we would eventually see side effects at that dose. Because as I said, the only data that I know of is that University of Tennessee group where they did have some dogs that survived more than a year and continued to take the drug. Those dogs, you know, as I said, had had hemangiosarcoma of the spleen, they'd had surgeries. You know, they were very sick coming into that study. And so I don't know even if there were mild side effects that you would really be able to tease that apart from everything else that's going on.

With those dogs, there are other larger mammal studies that are going on. Correct.

With rapamycin specifically. So there are. Well, so there are research studies in the context of aging in marmosets. They're very small and very early, but those are being done at the University of Texas.

Right.

San Antonio. Yeah, Texas. San Antonio. The Bar Shop Institute. And then there are a variety of cancer studies in dogs with rapamycin. So there's a large study of rapamycin for osteosarcoma in dogs. And then there are a few smaller clinical trials, but I don't know of any other large animal studies in the context of aging.

Anti aging.

Yeah.

Now, I want to obviously come back to the anti aging in the dogs, but on the cancer topic, there are some data that are actually suggesting that the increase in autophagy that one might see with rapamycin, which one would expect to see at least with MTORC1 inhibition, might be paradoxically not ideal in the active setting of cancer.

Right.

So I guess my first question is, are you swayed by those data? And if so, what would be the teleologic explanation?

Yeah, so I'll say I'm not swayed much. I think that those kind of data are important to be aware of. I think one of the real challenges in the autophagy field is that not.

Everybody, besides the Fact that we can't measure it easily, that's gonna be one of them.

Yeah. Beside the fact that we can't. We don't really know how to measure it. I think as a community, we really don't know what we mean when we say that autophagy is increased or decreased. Different people use different markers or measures for autophagy. So my view is that one of the things that can happen, not just for cancers, but for lots of different pathologies, is one of the ways that cells try to deal with many different forms of stress, in particular protein misfolding, but also other forms of stress, is to turn up autophagy. So I think that autophagy, some markers of autophagy going up can be a response to a pathological condition. Also, what often happens is that response of turning up autophagy does not lead to productive autophagy. So you actually get an accumulation of autophagosomes because they don't ever make it all the way through the process. So depending on how you're measuring autophagy, what you really might be detecting is a block at the late stages of autophagy, and what you're seeing is you're measuring a backlog.

That's right.

So it's not necessarily the case that more autophagy is bad in that context. It's the first failure to actually bring it through to completion. And again, I don't have a lot of data to support this. My intuition is that, at least for some diseases, one of the things that rapamycin does, and I don't really understand the molecular biology here, it seems to alleviate that block. So you actually get productive autophagy working again and again. I don't know exactly how that works, but that's what it seems to me is happening. And so we have seen some evidence for this in mitochondrial disease in the brain, where if we look in the brain, we can see these sort of massive autophagosomes that are trying to digest mitochondria that can't do it while the disease is progressing. And somehow rapamycin fixes that. So I think you have to be cautious in interpreting an increase in autophagic markers in a disease as necessarily meaning that increased autophagy is causing or contributing to that disease. And it could be the case that depending on how you activate autophagy, it could be detrimental or it could be beneficial.

And so there's really two different questions. The first would be, if you take a patient with cancer and you inhibit mtor, is it not helpful? Because the Tumor has already evolved so much to be outside of mtor's purview, or is it. It's actually harmful. And that's of course, separate from the option that could be helpful.

Right. So my understanding of the clinical and the literature in humans is that for most cancers, once it's reached the point of diagnosis, that rapamycin is disappointing in its effectiveness, it's not particularly effective. That's not true for all cancers. But for most cancers, it has not been as effective as you might expect. Given that we know that activation of mtor is common when you get high proliferation. And then turning down mtor should stop.

That, turn off a proliferative cell.

So I think you're probably right that at least part of the story is that one of the steps in the progression to cancer is evolving to ignore that signal, the break of turning down mtor. So rapamycin may not be effective there. I think it's a complicated system though, because the effects of rapamycin on the immune system could have beneficial effects in terms of cancer or detrimental effects. So we know that immune surveillance is probably the most important anti cancer mechanism, or certainly one of the most important anti cancer mechanisms. And we know that immune function goes down with age. That's probably one of the reasons why most cancers are age related. So if you can boost age related immune function with rapamycin, enhance immune surveillance, that's going to have a potent anti cancer mechanism. Again, this is my guess. My guess is that's why we see in the studies in mice that cancers are pushed back during aging by rapamycin. On the other hand, if the dose of rapamycin is high enough that you're actually inhibiting immune function, that could be.

That could promote cancers.

And there's not a lot of data yet. So we did one study in my lab where we gave mice, I think it's the highest dose that's ever been given in the context of an aging study. This was a daily injection of 8 milligrams per kilogram.

So as we call it the party dose.

Yeah, right, right. And so this was a study where we only gave the mice rapamycin for three months. So this was from 20 to 23 months. And then we stopped the treatment. And what was interesting there was we got completely different effects in male mice versus female mice. The male mice lived 60% longer after the end of treatment. They had better muscle function, they got less cancer. The female mice had no difference in lifespan. The mice that got rapamycin or didn't get rapamycin but they died with, I want to say from. But it's hard to say for sure what a mouse dies from. They died with very different types of cancers. So the female mice that had gotten this high dose of rapamycin for three months all had aggressive hematopoietic cancers, whereas about, I think it was about 30 or 40% of the vehicle treated mice. So in black six, that's not an uncommon cancer to get. But none of the rapamycin treated mice had non hematopoietic cancers, whereas like 60% of the mice that didn't get rapamycin.

Now, the 2009 study that kicked all this off actually showed a greater survival benefit in the female mice, didn't it?

That's right. So I think, and again, this is a guess because I don't actually have the data to back it up. My guess is that because we pushed the dose so high, we might have actually taken it too far in the female. So one school of thought is that female mice, at least we don't know if this is true in any other organism. Female mice are more sensitive to rapamycin and that could either be that they don't clear the drug as quickly or that for whatever reason, in female mice, the same amount of rapamycin has a greater MTOR inhibitory effect. But that's one school of thought and I kind of think that's right. So at lower doses of the drug, you see a bigger lifespan benefit in females than in males.

Did you repeat that experiment at like 4 mgs per kg or something different?

We haven't. We haven't. We should. So we did. Do we just need an infinite pool of money to do all of these.

Like to just answer all these questions.

Figure out the most important questions? Yeah, and I think the dose response is really important. We did do a lower dose for three months as well. And there we saw increases in lifespan in both males and females, roughly the same magnitude. So it was, that dose was nine times higher than what the ITP tested.

Wow.

So one of the things that's interesting though is as you go higher in dose, so three times higher than what they originally tested, the females still live a little bit longer, but the difference between males and females, the gap has closed quite a bit. So I think that females, for whatever reason, at a given concentration of rapamycin are just more affected by that amount of the drug. And I think what we did in our high dose study is we just pushed it a little too far. We pushed it to the point where rapamycin did something probably to the immune system that allowed these immune cancers to escape surveillance or become hyper proliferative. And again, I'm not a cancer biologist, I'm not an immunologist, so I don't have a good feel for what the mechanism is. I can tell you what the observation is and that's that all of those animals had aggressive hematopoietic cancers when they got this. Three months of rapamycin, just out of.

Curiosity, more B cell or T cell, do you recall?

I don't recall. It's in the paper. We could look it up because there's.

An opportunity here to do the reverse, right? I mean, there's an opportunity to take. Right now we're seeing just an unbelievable amount of activity in adoptive cell therapy or even when you just talk about checkpoint inhibitors and things like that, it makes you wonder, are there ways to make these things better? Maybe the checkpoint's the wrong example because you might get more autoimm. But certainly when you talk about adoptive cell therapy, anything that could boost either CD8 function or inhibit the regs or something, there might be ways. It almost makes you wonder if using rapamycin in a different manner in combination with immune based therapies might make more sense.

Yeah, no, I think there's a lot that could be done there for sure. Part of the reason why we haven't explored this in more detail, one reason is again, as I said, I'm not a cancer biologist, so it's not. That's not the thing I'm most interested in. I think it's really interesting biology, but it's not the thing I'm most interested in. But I also feel like, because the dose that we gave was so high, that again, thinking translationally about rapamycin as a drug in the context of aging, my feeling is that what we've uncovered here is not going to be relevant at the doses that we would think about giving to dogs. So that's why I haven't really spent a lot of my time trying to figure out what's going on there. But I think certainly in the context of cancer immune therapies, I think we do need to think a little bit more about how effective those kinds of therapies are going to be in the elderly. And maybe something like rapamycin could help, could actually enhance the ability of those therapies.

I mean, this question you posed when David Sabatini, Tim Ferriss and Navchandel and I were in Easter Island a year ago, over a year ago, this might have been our Favorite mealtime discussion, which is what best explains the increase in cancer incidence with age being. In other words, would the primary driver be the reduction in immune surveillance or the length of time to accumulate mutations or the frequency of mutations? Like, I mean, it's not an obvious answer.

And I don't think it has to be just one.

No, exactly. It's obvious.

I think all those things likely to.

Be all of them.

Yeah. Yeah. I certainly over the last few years have come to think that the decline in immune function is. It's certainly more important than I had initially thought. That's my.

I mean, I secretly want that to be the biggest driver because I think we have a better chance to control that than some of the other ones.

And I think it probably is. That would be my guess. And I also think it kind of makes sense that if you have an immune system that's functioning the way it's supposed to, you can actually deal with the mutation accumulation because your immune system is going to clear those before they become problems.

So now let's go back to the anti aging thesis. Right. Which is we're going to take healthy dogs, eventually healthy people. We want to reduce the rate of decline is probably the best way to think about it. Right. So we have a deterioration, that's the way I used to think in organ function. Okay. All right.

One of the things that's been subject to surprising to me again over the last few years is the different ways that rapamycin not only seems to delay the decline, but it seems to make things better. There clearly seems to be in at least some organs, a rejuvenating function. And I suspect that's mostly stem cell mediated. But again, the mechanisms haven't been worked out yet. So we've already talked about immune function. You can take an old immune system and make it work more like a young immune system. We've talked about cardiac function. You can take an old heart, you make it work more like a young heart. There's some evidence from David's lab that intestinal stem cells can be rejuvenated by rapamycin. We've recently published that alveolar bone levels, so in the mouth, the bone around the teeth can be rejuvenated back to a more youthful level by short term treatment with rapamycin. So there are now multiple different places in the body, at least in mice, where you actually see functional improvements back to a more youthful state. And so I don't think rapamycin is going to do that for everything, but at least for tissues and organs where Stem cell senescence plays a big role. I suspect that rapamycin can have not just an effect on delaying declines, but actually bring things partially back towards a more youthful functional state.

What's the best available evidence for that centrally in the cns?

Hmm, that's a good question. So there are studies on cognitive function in mice showing that you can improve cognitive function in aged animals. I believe at least one of those started the treatment late in life and saw improvements in cognitive function. And then in all of the major Alzheimer's disease mouse models, the literature's a little bit mixed. There's at least evidence that you can wait until the pathology of the disease has set in. You see the A beta accumulated, you see the functional deficits in terms of cognitive function. You can start the treatment and you can improve things.

And on postmortem, are you seeing an actual reduction in amyloid? Yeah. You are.

So we haven't done this. This is the work of several other labs in the AD area. Yes. So, again, the data's a little bit mixed. So there are a couple of papers out there where they see that you can get really robust benefits if you start rapamycin treatment early, but they didn't see benefits in the AD models when you started late. And then there are studies that did see declines in aggregation, increased autophagy, and functional improvements. It would be startling, it would be.

Staggering, if you could take an animal, and ultimately, of course, a human who's already accumulating AB and tau.

Yeah.

And even just halting that is a big deal. There's one drug that the FDA's approved now.

You're going right into my biggest pet peeve right now, which is why there hasn't been or isn't a current rapamycin trial for Alzheimer's disease. But I think you're right. And again, three years ago, if somebody had asked me, will rapamycin, is it likely to have any benefit in somebody who's been diagnosed with ad? I would have probably said no. I come around to thinking that there's at least a reasonable chance that it can not just halt progression, but it could actually make.

Well, especially if you. One of our colleagues is a neurologist here named Richard Isaacson, and he runs the largest Alzheimer's prevention clinic in the country at Cornell. You know, Richard's thesis, which I think makes a ton of sense, is, again, I think other people share this view, is you want to catch people while they just have the first signs of mild cognitive impairment. Sure, absolutely. And your ability to actually impact them is Enormous. And so the question is, why aren't those people being considered for clinical trials when we already know that these other agents aren't really doing anything right?

Yeah, I agree. I think that if I was going to design a clinical trial for Alzheimer's disease or dementia with rapamycin that I thought had the best chance of success, that would be the target population. Those trials are harder to do in some ways because they're longer.

Right.

And not everybody moves from MCI to full blown AD at the same rate. And we don't have, at least my understanding is we still really don't have great predictive biomarkers of how fast that's going to happen in an individual. But that would be the study that would have the best chance of working. Having said that, I still think there's a decent chance in somebody who's already gotten to the point where they will be diagnosed as having Alzheimer's disease. Serious functional deficits. There's a chance that rapamycin could make things better. Now I get the practical reasons for why people don't want to try a risky clinical trial. It's expensive. If you fail, you know, then your drug gets a bad reputation, all of that. So I understand why people are hesitant to do that trial. I think there's actually a pretty good chance it would help.

I think you can combine it. I mean, I think, you know, Richard often talks about, and others do as well, that one of our failures in Alzheimer's is we consider it a single disease.

I agree.

It's as naive as saying John has cancer. Oh, well, gosh, that's the end of his life. Well, don't you want to know what kind of cancer first or maybe what mutation? So similarly, but broadly speaking, and this is a gross oversimplification, if you consider the metabolic version of Alzheimer's disease, the vascular version of Alzheimer's disease, and then the sort of toxin clearance impaired version of the disease. To me, I'm generally most optimistic about the metabolic one. So the variant of this disease that seems to be mostly due to a failure of energy metabolism in the brain, that also strikes me as the one that's most amenable to systemic therapies as well.

Right.

If you improve insulin sensitivity, if you improve glucose disposal, if you reduce hypercorticolemia, you can actually, through nutrition, exercise, sleep, a number of other things start to modulate that. That strikes me as the one where you want to at least take your first shot at adding rapa.

I think you're probably right. Although again with rapamycin because we know that it is effective at, for example, turning up autophagy. It might also be.

It might work in the harder ones. That's a harder one.

I agree. I tend to agree with you. I think that's a good point. I also want to add that I actually think the biggest problem with the way that the scientific community has thought about Alzheimer's disease, aside from considering it to be one disease, is not really recognizing that it's a disease of aging. I really think that one of the reasons why the pre clinical research has been disappointing at developing therapies for Alzheimer's disease is because very rarely have people approached that from the perspective that this is a disease of aging. And so something that can affect the mouse models of Alzheimer's disease, when we create this disease in young mice, may not work the same way in an aged person or an aged animal. And that's one of the things that also makes me optimistic about rapamycin is we already know that it hits the hallmarks of aging and it also seems to be effective in these Alzheimer's disease models. So that makes me think that it's acting at a sort of a more fundamental level. Fundamental level to target the molecular causes of this disease.

Well, especially look, I mean, if you can regenerate a cardiac myocyte from its stem cell, it's not an impossible thought that you could regenerate neurologic stem cells.

Absolutely. And this is again an area Which.

I mean, 20 years ago we would have said that's impossible, it's metaphysically impossible.

Right.

Going back to the dogs for a moment, again, I'm not familiar with dog literature. What is the fasting literature look like in dogs?

There's not a lot that I'm aware of either, and I think that kind of makes sense. So, you know, first of all, you have to differentiate the literature in laboratory colonies from companion dogs.

Companion dogs.

Right. There's probably not much in companion dogs I don't know of true fasting experiments in like bee.

So we can't use like the fasted versions as proxies for what we would hope to see on the RAPA dogs. Which is the way they sort of did it in mice. Right. They sort of said, well, you know, we know that if you calorically restrict this mouse, this strain of mouse under this degree of caloric restriction can expect this much of a longevity boost. And oh, lo and behold, rapamycin is probably even better than that.

Right. Although from a metabolic perspective, I think it's still unclear even in mice whether Rapamycin and caloric restriction are working through the same mechanism. And this is actually another area where there haven't been a lot of good experiments done. So there's a portion of the field that argues strongly that caloric restriction and rapamycin are completely different, which to my mind is absurd. I mean, we know that one of the main things that caloric restriction does is it inhibits mtor and we know that rapamycin inhibits mtor. So they're not fundamentally different. So I think they are overlapping but distinct. Not everything that caloric restriction does is going to be mimicked by rapamycin and vice versa. Having said that, when you look at the gene expression profile or the metabolic profile, they don't look all that similar, at least at the low doses of rapamycin. And so I think it's a little bit unclear. You're right. Lifespan. They both extend lifespan. Rapamycin might actually extend lifespan across a broader background than caloric restriction. But when you get beyond that, I think it's still an unknown whether there are, say, are there metabolic signatures that are common to both that might therefore be more likely to be causal? I think we don't know the answer.

So what you're really getting at is probably the next thought I had subconsciously, which is, could we use calorically restricted animals to develop a metabolic signature as the gold standard for how we would then titrate rapamycin both in dose and frequency?

I don't know of any evidence to support that.

That would be a stretch.

Yeah, I mean, it might be possible, but there just isn't much good data out there at this point. I do feel like if we want to put the resources into trying to identify predictive signatures, which I absolutely think we should do, I think the metabolome is probably the place to go and the serum metabolome makes a lot of sense. So that would be where I would look. Nobody's really done it. Well, part of the problem is the data that's out there for rapamycin is again all at that very low dose that the ITP tested originally. We know that's suboptimal for lifespan. It's quite likely that the effects that the lowest dose of rapamycin are having on metabolism are going to be relatively modest compared to higher doses. So the changes might be there, but they might be so subtle that you're not going to detect them in a sort of high throughput screening approach. So I think there's a lot we don't know, but that would be where I would put my effort in trying to identify serum metabolomic signature of rapamycin that we could then correlate with the effects on function in a variety of different tissues as well as lifestyle.

Yeah. Because I mean, I think as wonderful as it would be to just wave magic wands and have biopsies, if you tissue, it's just not going to be. I mean, we're not going to take cardiac biopsies.

Yeah. Right. So if you're looking for predictive signatures, you can do those experiments initially in mice, but you really have to think about is this something that's reasonable to do in a person or a dog?

Have you looked at the gut biome in the dog at all?

A little bit. So we did a study in mice. This was that study I referred to previously, where we treated them for three months with either the high, high dose or the. The high, but not. And we did look at the fecal microbiome and we saw quite dramatic changes there, some of which are interesting. And so that's an area that we would love to pursue. I tried to get a grant to do that and so far haven't been successful through nih, but it hasn't been.

Done in dogs yet.

Has not been done in dogs. So we have a little bit of preliminary data from our phase one study and we're seeing changes in the microbiome there as well. So far, the data that we've got in dogs is not the sort of comprehensive metagenomic approach where we sequence everything that's there. It's more of a standard veterinary approach where they have sort of broad classifications of different types of microbes and there are definitely changes with rapamycin. It looks, it's very early, but it looks as though, at least in dogs that have a bad microbiome, a dysbiosis in their microbiome, which can be defined clinically from this test. There were two dogs that we've looked at that started out with a bad microbiome. They were in the rapamycin group. They were better by the end of the study. Obviously don't know if that's meaningful or not. So there are changes in the dogs, but it's really too early for us to know whether they look like the mice and. Or whether that is going to either be predictive or potentially play a role in some of the effects of rapamycin. It is definitely the case, though, in both dogs and mice, and I'm sure this will be true in people as well, that rapamycin has a large effect on the composition of the gut microbiome. I suspect that will also be true for other compartments in the body like the oral microbiome, maybe even the skin microbiome. Nobody's looked yet. So there's a lot to be done there. I also don't necessarily think that's going to be unique to rapamycin. I think that lots of drugs that we take.

Yeah, food will change and Absolutely.

Diet.

And it's not clear if that's the effect or the cause of it.

Yeah. So one of the reasons why I think that there's reason to think that at least some of the changes in the microbiome could be caused for some of the effects of rapamycin is that one of the things we saw in our mouse study was a pretty profound increase in bacterium called segmented filamentous bacteria or SFB in the rapamycin treated mice. It turns out if you look in the literature, there are links between SFB and diabetes and obesity, and also between SFB and T helper cell maturation. So it could be the case that changes that rapamycin is having on this specific bacterium as well as other bacteria are then having effects both on potentially nutrient utilization and uptake, but also direct effects on, say, immune function. I mean, the intestine is an important immune compartment. The bacteria are physically right there. These SFB actually form filaments directly associated with the intestinal cells. So it certainly could be the case that there are signals being sent back and forth that rapamycin is modifying through modification of the composition of the microbiome and that that's affecting immune function, adiposity, all sorts of different possibilities. So like I said, that's something we're really interested in testing.

There's like an infinite number of things I want to know. There's this company out there that's looking at duodenal ablation to ameliorate diabetes. And the data are actually really interesting. So interesting, in fact, that when I first saw them, I thought, this has gotta be nonsense. What's not clear is the durability and the economics of it. But just from a scientific standpoint, they're doing these ablations of the duodenal mucosa and they're seeing like immediate step function changes in insulin sensitivity, arguing in fact, that this may be the mechanism by which the Roux en y gastric bypass is ameliorating type 2 diabetes. It's basically taking this dysfunctional duodenum out of the loop and just saying we're going jejunum to jejunum, or you know, stomach to jejunum to jejunum, basically, it's really interesting.

So this is another area that I've gotten interested in and we haven't really dove into it yet much, but I think there's clearly literature growing in all of the model organisms that with aging there is a decline in intestinal barrier function and that at least in flies, that seems to be causal for death. So in other words, it's strongly correlated. And there are ways, every way that extends lifespan also seems to improve this intestinal barrier dysfunction. And I've been thinking, and we have again, a little bit of data suggesting that rapamycin might actually have an effect on this age related decline in intestinal barrier function. And why would a loss of intestinal barrier function be important? Well, I mean, one thing that happens, we know this happens, is that you tend to see an increased level of microbial proteins and DNA in the circulatory system with age. That causes an inflammatory response that may contribute to the sort of systemic increase in inflammation that we see during aging. So the loss of intestinal barrier function may actually drive to some extent the inflammaging, the increase in inflammation with aging, or at least contribute to it. And so anything that you can do that is going to improve that will potentially have an effect on systemic inflammation. And that again could be another way that rapamycin is sort of impacting the entire body. In addition to the effects of rapamycin when it gets to a cell and inhibits MTOR in that cell, and whether that's through changes in the microbiome or changes in intestinal stem cell, like Sabatini has shown. Right. There's lots of possible ways that it could be working. But I do believe that this decline in intestinal barrier function with age probably is contributing to this sort of increase in systemic inflammation that we see during aging. And it's very clear that that happens in people and in non human primates as well during aging.

Now, talking about people again, I don't hold out much hope that there's going to be an anti aging trial in humans using rapamycin or a rapalog. Watching how much difficulty it is to even try to get that study done with metformin, which I mean is about the most inert. It's like you might as well just do it with drinking water. We're gonna randomize you to seltzer versus flat water here. So the real question is, for the people who want to be on the tip of the spear, you're not gonna have gold plated stamped rct. You're going to have to triangulate from everything else. Many roads point to your work and more importantly, what your follow up work will look like. So again, I realize that to try to secure funding to do this from NIH is slightly more complicated than trying to get bipartisan support on health care. But if we took the economics out of the equation, if there was a 10 to 20 million dollars pool that the National Institutes of Aging said, you know, Dr. Kaeberlin, we want to, we want the definitive work on how to extend the life of dogs, knowing that that's probably the best thing we're going to get towards humans. What do those experiments look like?

Right. So the study that we want to do is a five year study with rapamycin. And certainly this is scalable. Right. So what we have designed is a study with rapamycin. There are other interventions coming down the pipeline that I think have the potential to be as effective as rapamycin. So this doesn't necessarily, as long as you can do it safely in pet dogs, you can test any intervention. And one of the things that I am now convinced is owners are enthusiastic about participating in these kinds of studies. We've had more than 6,000 people sign up through our website with no advertising at all to participate in the rapamycin trial.

In human clinical trials, which I'm more with familiar, familiar with recruiting cost is an enormous amount.

It doesn't cost anything.

Yeah, you get free recruiting.

So having said that, the study we would like to do is a five year study in dogs starting treatment at middle age. So again, the dogs would come in at least six years old. We might push that up to seven or eight years old and there would probably be a weight limiter like I talked about before, because again, big dogs age faster. So let's just say 40 pounds, 6 years old, at least 6 years old. So there will be dogs anywhere from 6 to 10 or 11, bring in 450, 500 dogs, or enough dogs to get 450 all the way through the study. And look at not just lifespan, but lifespan is actually a really important metric here. Right. Lifespan is certainly still the gold standard in the aging field. If we want to convince the scientific community that this is affecting aging, it's darn well better increase lifespan. It's also important to owners for obvious reasons. And I think because as we talked about, euthanasia is really what most dogs die from because the dog gets sick enough that the owner and the veterinarian Decide that it's time to put the dog down. I think lifespan is actually a really good metric of health span in dogs because most of the time that's not an easy decision. Right. Owners are not going to put their dog down, usually especially owners who want to participate in a study like this, unless that dog is really sick. So I actually think lifespan is really maybe more important in dogs as an outcome measure than it is in mice. So lifespan is one of the key endpoints that we want to look at. And then we want to look as broadly as we can at functional measures of aging that we know are important in dogs. And so this goes way back to what we were talking about before. What do dogs get sick with as they get older? So heart function we will definitely look at in part because that's what our preliminary data.

Yeah, you showed that with a handful.

Of dogs we have good evidence that we can detect and expect to detect improvements in cardiac function activity. So one of the nice things about dogs is you can put a GPS tracker on their collar and get very quantitative measures of activity. As the sensor technology improves, it might be feasible to use a microchip instead of a GPS tracker on the collar to also get some physiological.

Or just throw a Fitbit on their wrist.

Yeah, right. Well, I mean, that's really what the tracker's gonna call it. But it would be nice to get some physiological measures in sort of continuous real time if we can. So far, the sensor technology isn't quite there, at least that's my understanding. But we'll at least get activity and that'll give us a measure. Both of things like arthritis. So dogs that have arthritis are going to be less active and also muscle function and also how well are they feeling? Again, a dog is more likely to be active if it feels well. Now obviously that's confounded a little bit by whether the owner takes the dog for a walk, but I still think total activity is an important thing.

And is this a three group study in your mind?

So the study that we're designing now is three groups. It doesn't have to be, but the three groups are a placebo group, short term group, so six month or year long treatment, and then the continuous treatment group. The reason for doing that again comes back to the mouse data in part because we, as I mentioned, we've published that a short term treatment in mice is enough to give you large benefits on lifespan and at least some measures of health. And also because again, as we're thinking about ultimately bringing this to people, it's Easier to envision transient or intermittent treatment than it is a continuous for the rest of your life sort of treatment.

So it'd be single dosing.

Again, that's the way we have the study designed. Now I can see a rationale for doing three months on, three months off, or some variation on that. We felt that the simplest thing to do first. So, you know, it's always a balance between getting as much information as you can from a study like this and doing things that are going to be where the complexity isn't so great that it.

Yeah. Your power analysis could give you a study.

That's right. So the design that we're working with now is one year on and then the rest of the time off. And so those. So we'll get.

So you have one group that's five years on, one group that's one on, four off, and then placebo.

That's right.

Blinded across the board.

Yeah. It's all going to be a randomized double blind trial. Right. In addition to lifespan and heart function and activity, we'll track cancer incidence.

Probably kidney function.

Kidney function. Get routine blood chemistry on the dogs probably every six months. We'll ask the owners to bring their dogs in, get echocardiograms, collect serum for metabolomics and feces for microbiome.

So what would that study that you just described cost if you could complete? So let's say you had to screen 600 dogs to complete. 450, which would actually be pretty good attrition.

So it would run about 5 million. That's about the budget for that study. Some of that depends on what we can ultimately get the rapamycin for.

Because right now the street value is.

About the lowest we've been able to find. About $7amilligram. Yeah. Now it may be possible if we have a large study that we can identify a supplier that would cut us a deal for less. And the drugs are actually a pretty big part of the cost of the budget. Yeah, the veterinary costs are the other large expenditure. Again, the nice thing about companion dogs, it's unlike a study in mice in the lab is they live with their owners. We don't pay cage costs and things like that. The other major costs are going to.

Be for the analysis.

The analysis. And also we need people to be able to communicate with the owners. Retention will be important. I don't think it's going to be as hard as it is for some clinical trials just based on our experience, even though it was a short term trial. The owners that came into the short term trial, they were extremely.

This will be easier than communicating with patients in a trial, I suspect.

I think so, yeah. And there's actually. I mean, it's kind of funny, but there's actually data that dog owners are more likely to give their dog a prescription medication than they are to take.

Their own prescription medication. I would not doubt that for a second.

So, yeah. So the donors that come into this study are highly motivated, and some of them are extremely disappointed after the study ends when they find out their dog was in the placebo group. So. But there is a communication component to this where we have to keep the owners engaged, maintain communication. We'll be sending out regular surveys, but needs to go beyond the surveys.

Now, would these all have to be dogs that live in the Northwest? They'd have to be able to come.

In fact, they almost certainly would not be. So the way that we're planning the study now is that we will partner with five to seven veterinary schools around the United States. Oh, that's great. And that actually, there's lots of reasons why that's. Well, diversity alone is great, but veterinary cardiologists. There's not a huge population of veterinary cardiologists out there. So if we were to try and do a study like this in the Seattle area, I don't think that we have enough veterinary cardiologists to actually do just the cardiology part of the study. So fortunately, veterinarians are very enthusiastic about participating in projects like this. And we have collaborators lined up at the vet schools around the country. So it will probably be five or seven sites. Obviously, all of those sites have to have veterinary cardiology, but almost any major veterinary school is going to have that. And we prefer to work with veterinary schools that are in a suburban or urban area. Some veterinary schools, like our school in the state of Washington, is all the way on the other side of the state from Seattle. It's. It's kind of out in the middle of nowhere. So that makes it harder to get owners to actually bring their dogs there. Yeah. So our lead veterinarian is at Texas A and M Veterinary College. And so she would be the head clinical person on the study. And that would probably be the site that the other veterinary sites coordinate with.

Yeah. Well, Matt, I could sit here and have this discussion for another two hours.

It's fun stuff to talk about.

It is. And I really appreciate your time and your insights. And I think the work you're doing is certainly what I would consider to be among the bodies of work that are at that tip of the spear, because I guess I don't have a lot of hope we're gonna get the answer to this question directly. I think it's going to be an indirect triangulation of data. And I think to be able to do this in companion dogs that live in our environment is going to be a really important thing. So let's see if we can get that study done.

Yeah, I also want to. I mean, I think it's also important to at least know to the impact of a study like this would have on public perception. Right. I mean, I think in the absence of any data and then the little bit of data we got from the phase one study, the amount of media attention that we've got.

Yeah, I've heard you on NPR talking.

About this with Terry Gross has been huge. And so I tend to agree with you that it's going to be challenging to generate enthusiasm for a double blind placebo controlled clinical trial of rapamycin for healthy aging in people. Challenging is probably not even a strong enough word. But I think we do need to, or we should at least not underestimate the potential impact if we're successful at accomplishing this in people's pets that that will have on public perception as well as perception among the broader scientific community. I do feel like the field of aging research still has a bit of a reputation problem among the broader scientific community. Part of that is historical. Part of that is because there are some fringe elements that get a lot of attention but that aren't scientifically credible. I think that actually being able to show in dogs living in the human environment that we can modify aging will have an impact not just on the public in terms of, I'm sure we'll get lots of media attention, but also among scientists who might actually say, okay, aging research has arrived. I think that's happening already. I think the TAME trial has actually been mostly a positive in that sense. Or the proposed TAME trial, I should say, but I think we still have.

For the listener is the targeting aging with metformin.

Yeah. Which is. I mean, I support that study. I think that it's probably the right first study in this area because as you've already said, metformin, we know that it's very safe, at least as far as a drug you might consider for a study like this. It's very safe. There's very good human data suggesting that diabetics taking metformin not only have less diabetes, but they have fewer other age related diseases. So I think the human data is pretty Compelling. The downside to metformin and I think one of the reasons why it has been a struggle to get that study funded is that I think there's a perception that we already know about metformin. It's not going to be completely surprising given the literature that's out there. If it does have relatively small effects on other age related diseases. And then I also think it probably isn't going to have that large of an effect. I could be wrong. I hope I'm wrong. But again, my view of the literature that's out there is that metformin probably has modest effects, but they're not going to be 20% increase in lifespan and rejuvenation of heart function and immune function.

Yeah, I agree.

So I think that that's probably the downside to that study. But again, because we're also battling this perception of resistance to the potential for side effects. When you're talking about treating healthy elderly people, that's probably the right way to design this first study. The other thing about the TAME trial though is it's not being done in healthy elderly people. Right. The people that they're enrolling have to have at least one age related disease and it can't be diabetes. So even that is really not the gold standard study that we'd all like to see. Whether it's metformin or rapamycin or, or something else, we've got some work to do to get to where we can actually do that study. And maybe the path forward is, as you said, individuals who are willing to come together and do these sort of self experiments. If you can do it in a rigorous way where you're actually measuring the things that you're doing, there needs to.

Be a model system that allows it so that if you and I and Bob and Rick and John do it, we're.

The downside to that kind of a model is that it's still going to be hard to convince just the scientific community the way that it is. It's much harder to convince them that it's real. Having said that, if the effects are robust and you see it over and over and over in multiple people, we'll get there eventually. So that may be the path that we end up taking.

I'm not betting on that path. I'm betting on the path that you're describing as being the shortest distance between these two points.

And I hope that as we start to do more of these studies in dogs and also the preclinical stuff in mice, as we start to find functional measures that are improved over a relatively short timeframe that people will start to do some of these clinical trials that are feasible in people. I mean, you could. I mean, Novartis has already done it, right? You can do a clinical trial where you treat healthy elderly people for six weeks, eight weeks, ten weeks, and look at a functional outcome. And so if it's the case that rapamycin rejuvenates immune function and rejuvenates cardiac function and delays or restores alveolar bone levels in the mouth, right. Those are clinical endpoints that are impactful and could be studied in a short clinical trial. So we may get a few of those that will kind of build this body of evidence that rapamycin is having a similar effect in people. Again, the hard part is there's not a lot of money in rapamycin, so I don't know who's going to fund those trials. That's the challenge. So we need to identify. So either it's going to be rapamycin derivatives that are under patent like Novartis has developed, and now restorebio is further developing, or it's going to be alternative funding sources, whether that's foundations or wealthy individuals who recognize the potential impact of this work and are willing to fund a clinical trial to actually start to look at some of this.

Well, Matt, thanks again. This was super interesting and best of luck with your continued work.

Thank you.

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