Gladden longevity — Episode #23

Speaker 1: Welcome to The Gladden Longevity Podcast with Dr. Jeffrey Gladden, MD, FACC, founder and CEO Gladden Longevity. On this show we want to answer three questions for you, how good can we be? How do we make 100 the new 30? And how do we live well beyond 120? We want to help you optimize your longevity, health and human performance with impactful and actionable information. Now, here's today's episode of The Gladden Longevity podcast. The Gladden Longevity podcast is provided for informational purposes only. It does not constitute medical advice.

This content is not intended to be a substitute for professional medical advice, diagnosis or treatment. Always seek the advice of a physician or other qualified health provider with any questions you may have regarding a medical condition. The use of any information and materials linked to this podcast is at the listener's own risk.

Dr. Jeffrey Gladden: Today on The Gladden Longevity podcast, we're going to go deep on DNA methylation and epigenetic aging. We're going to actually really demystify this for you in terms of what causes it, how to measure it, what impacts it, how do we change it for the better? I think you're going to find this conversation with Ryan Smith, really fascinating. I've known Ryan for about four or five years. He's a graduate of Transylvania University in Kentucky. He did two years of medical school at the University of Kentucky. When he started his clinical rotations realized that he didn't really want to be a doctor.

From there he got involved with peptides and opening Tailor Made Pharmacy. He left them in 2020, and he's now been working with a new company of his called, TruDiagnostic, which is a clear certified lab that basically focuses on measuring epigenetic age. I think you're going to be fascinated to see how this has grown from what you've understood about DNA methylation and epigenetic age in the past. I think you're going to love this conversation.

Welcome everybody to this edition of The Gladden Longevity podcast. As you know I'm here with Ryan Smith. Ryan and I have known each other for what? Five years now? Four years? Five maybe? Something like that. We've had the opportunity to work together in a couple of different settings actually. I'm excited for this show because Ryan is working in an area that fits in nicely with our circle of longevity. One of the key metrics in the mosaic of ages that we measure are epigenetic ages. It turns out that epigenetic ages have expanded both in terms of the number of epigenetic ages we can now measure for an individual as well as get a rate of aging. We can estimate telomere lengths. We can do more and more.

Ryan and I have been working on a couple small little research protocols together, and he's got some really interesting ideas about where epigenetics or DNA methylation can take us. But before we get into all that interesting stuff, Ryan, why don't you tell us a little bit about your vision for TruAge and really where you want to see this go. Give people maybe a bit of an overview.

Ryan Smith: To start, I think it's important to define TruAge and what we're doing there, which is generally our suite of aging related biomarkers. I think fundamentally our company was built on a belief that age itself is a disease process, which can be mitigated if you can properly manage it or measure it. And so that's what we've really tried to do, is to create the best suite of aging algorithms for physicians and patients to use to quantify their aging process. I think that in this area, aging will continue to have better and better biomarkers, which will be more informative about the processes which generally tend to degrade with that aging process.

But I think if there's one big takeaway for anyone listening, it's the potential for epigenetic methylation, even beyond just aging. That has definitely been one of the ways that it started, but now we're able to get surrogate biomarkers for many other things that are important to the health process. And so I think that with one simple test that uses just a couple drops of DNA and a couple drops of blood, we can get a lot of different biomarkers that can revolutionize health in a lot of different areas. We've definitely taken the lead in aging and will continue to have that as our focus, but as we generate more and more data and test more and more patients we're able to do a lot of other interesting things that are relevant to health as well.

Dr. Jeffrey Gladden: Let's back up for a second and define for the audience what DNA methylation is, how it works, how it integrates with the Horvath clock, other aging clocks, things like that. Maybe let's just talk a little bit about that for starters. And then we can start to move into some of the more sophisticated stuff.

Ryan Smith: Definitely. Well, epigenetic methylation, or just epigenics generally stands for above the genome. And so these are changes which happen to our genetics, which change and regulate their expression. Generally I like to say they're the on and off switches for our DNA expression, what DNA has turned on and what DNA has turned off is regulated by some of these epigenetic marks. There are two main epigenetic marks, which is generally the one that we look at, epigenetic methylation, which is generally that off switch, where these are attached to certain areas in the genome, which can prevent transcription.

Vice versa, we oftentimes see the DNA acetylation is that other big marker, which is oftentimes the on switch, where that basically just opens up your DNA from the proteins which store it and let the DNA be transcribed by the mechanisms which take our DNA and then make it into RNA, which then goes to peptides and proteins. That's sort of what-

Dr. Jeffrey Gladden: Just interrupt for a second. When you're measuring basically methylation patterns on the DNA, and people should understand that these methyl groups, these carbon with three hydrogens, they get added at certain points through life. They get subtracted at certain points through life. It's really, what's being measured here is the pattern of methylation, the pattern with which these groups are on the DNA, so to speak. And it correlates with the fact that the pattern of methylation, to Ryan's point, will determine what genes are suppressed. And the flip side of that coin of course, is which genes might be activated?

But that changes as we go through life. It changes as you go through puberty. It changes as you're a child. It changes, when you're in your 20s. And it also changes in conjunction with the aging process. Steve Horvath has shown that there's a methylation clock associated with this changing pattern of methylation that goes on that's really pretty much constant across all mamalian species, quite honestly. This is just to give the audience a sense, this is a very robust clock that is keeping track of the aging process, if you will. And it's probably a little bit of a chicken and an egg scenario here too, where it's actually the methylation pattern changes in response to the aging process, but then the methylation pattern also contributes to the aging process, which is really pretty fascinating.

And that being said, your company, TruAge, is measuring methylation patterns. I think you guys do a great job of that. Are you measuring the acetylation patterns on the histone proteins as well, or what's happening there?

Ryan Smith: We are in some of our clinical trials, but honestly it's a little bit more difficult, a little bit more expensive and significantly less data output. And so that makes really not ready or not as ready for primetime large scale investigations as epigenetic methylation. And even in epigenetic methylation, we are still, I would say at the very beginning of this type of technology. When Dr. Horvath first created his clock in 2013, they were able to look at right around 450,000 methylation sites at maximum. Now we're doing over 900,000 with each of our tests, but that's still only a fraction of the total amount that we could look at per every cell. There are over 29 million in each cell.

And so definitely one of the biggest, I would say breakthroughs in this technology will be continuing to scale this to larger degrees of information while keeping the price down and seeing the same type of evolution we saw in the genetic industry. We're only measuring epigenetic methylation, but at the same time this new technology is happening from a molecular standpoint, we're also seeing a lot of new technology applications for computer learning and artificial intelligence. And this allows us-

Dr. Jeffrey Gladden: Let me just ask, let me just interrupt you there, because if you're going to measure 900,000 genes, is it that much more difficult to scale up to two million, to four million? Does the accuracy of the test, the specificities, the sensitivity of the test improve that much or are we going from 98% to 98.3 to 99 to 99.1? Are we at that point on the curve where it's starting to really plateau in terms of the yield or where are we there in your sense?

Ryan Smith: Definitely. We are starting to plateau in terms of how much information is actually useful for the accuracy and precision of these algorithms. But with that being said, the advances are happening very, very slowly. By doubling it, we're not necessarily seeing a doubling of accuracy by nature. And so these things are incremental, but as we get more information on what genes are turned on and turned off, we get a little bit more information on the pathophysiology. So what is changing and why it's changing in order to give us insight. And that's one of I would say one of the biggest question marks surrounding these clocks is, I think you already mentioned that sometimes it's hard to separate them from disease, but oftentimes it's even hard to separate them from aging.

We don't know right now if they're causal or just correlated. We know these clocks are incredibly predictive of disease. We know that they're probably the best way to capture the aging process, which is the biggest risk factor for chronic disease and death. But at the same time, we don't know if this is a cause or just a correlated factor. And that's some of the things we're trying to answer as well.

Dr. Jeffrey Gladden: Yeah, no, it makes it quite fascinating. One of the other things that you alluded to was that testing more sites may be more expensive. Is that true? Does it take more reagent, more time or whatever? Are we hitting a plateau in terms of cost effectiveness? Is that what you're thinking as well? Tell me about that.

Ryan Smith: Definitely. We were absolutely hitting, I think a little bit of a plateau on cost effectiveness. This also happens from how we create the algorithms versus how we run them or report them. We choose 100,000 spots, even though a lot of these algorithms can use as little as 113 per every algorithm. And the reason we do that is because we still feel we're not nearly through the learning phase yet. And so we want to make sure we have a lot of different information that then we can report on in perpetuity. For us, we update reports, we just did a big update to increase the precision of these algorithms in early June.

And we will be doing that for several years to follow as the science gets better and making sure that we can continue to update those results as best we can for both our patients and [inaudible 00:10:49].

Dr. Jeffrey Gladden: Let me tell the audience why you should care about this. The reason you should really care about this is that what Ryan is really attempting to do, and I think it's admirable, is to actually be able to use the DNA methylation patterns to not only give us an idea of where we are in a biological timeline, so that we get a biological age, if you will. And actually we're all a mosaic of many biological ages, but that being said, this would be one of those ages. And even inside the methylation world, there are multiple ages. And we'll talk about that in a minute too.

But what he's talking about here and why you should really care about this is also because they are able to see with certain methylation patterns, different disease types, or predict disease types, and even start to predict things around mortality and things like that. It becomes more than just, my chronological age is 50, but my biological age is 42, therefore I'm terrific. There's much more information to be had from that. It's really, I think it's deceptive to take one number and compare it to your chronological age and think you've got it dialed in, because we really are mosaic of many things.

And to that end, what Ryan is doing is understanding that we're a mosaic of many different ages and trying to use DNA methylation to actually go after those different things that comprise that mosaic, to be able to predict it, utilizing DNA methylation. You're basically taking technology and also allowing yourself to start to understand disease processes, et cetera. I think that's really fascinating. I've never heard about that with any other DNA methylation company, really.

Ryan Smith: We're a little unique there because I think that like you, Dr. Gladden, we have a similar philosophy, where we know that epigenetic methylation is a great biomarker, but in and of itself it can't tell us everything. So what we really want to do is combine really what we call this multi-omic thought process, where we're gathering information on multiple layers of the body's function, and then training that to methylation, to see if with one test we can be able to put out many different things, which can give us an overall picture of a lot of the different bodies’ processes. We're not the only ones leading this, this idea of epigenetic methylation is growing very, very rapidly.

Dr. Jeffrey Gladden: Yeah, yeah, yeah. No, there's a lot of push in this direction. Everybody's learning from everybody at this point. But that being said, maybe you can fill us in on if somebody has a true age report, we've been using your testing for about a year now I think, maybe a little bit more. I can't remember. You've opened this up about a year ago to us, I think. Tell us a little bit about the reporting, what somebody can expect to get, because you've got the intrinsic age, the extrinsic age, the DunedinPACE age. Just walk us through a little bit if somebody gets this test done, what they might expect to learn.

Ryan Smith: Well, I would love to maybe tackle those reports. One of the things I want to do in order to tackle those reports is to explain a little bit of the evolution of these algorithms, how they first started and then where they are now. Because that makes a big difference. These first algorithms specifically, the one that Dr. Gladden Mentioned, with Dr. Horvath from 2013, that was really a hallmark paper. It was a clock which worked in almost any tissue and across mammal species. It was a really, really amazing clock, which I think Dr. Horvath might be considered for the Nobel prize for.

But that was trained against this idea of chronological age. These methylation marks were meant to predict someone's chronological age. And so that was a really big breakthrough. But the problem with that is the better that those clocks got, the more accurate that they got, the closer they got to your birthday. Right? Which is just something we can ask for. And so those clocks, those chronological age clocks have been passed over in favor of other types of clocks, which have been trained against other types of information. We call these now the second and third generation clocks. And so these clocks are trained to health phenotypes.

The things you might see if you're measuring these clinical values in patients, things like blood levels, things like Lucas telomere length, or lung functioning measurements or lipids and triglycerides or cholesterol, right? These are giving us more of a signal of health. They're not just being correlated to the chronological age, but they're correlated to those health outcomes that we see. And so with our reporting, we're definitely making a shift into that direction. That is very important because as you're doing this testing, you want to know what it's telling you and some of the limitations of it.

And so whenever we first unveiled our testing in July of 2020, we only had one report. And that was a chronologically trained clock that was still very useful and still incredibly accurate. However, now we're adding different reports. And so if you were to do our testing, you would get what we call this intrinsic and extrinsic age. Those were really the first reports that we offered. Both of those are chronologically trained clocks. The intrinsic tells you how old your body is fundamentally. It would give you an age and we would want that age to be as low as possible. If everyone in the world were to be seven years younger than their chronological age, that's when we would see a 50% reduction in morbidity and mortality risk.

And so that seven year age shelter is absolutely something that we would try to get all of our patients and consumers to really try and hit. In addition to that, we have the extrinsic age, which is the age of your immune system, which can give you an idea of how your immune system is aging. And with that, we do things like immune cell subsets, where we're able to tell you how many CD8 T cells, or how many CD4 T cells or neutrophils that you have as a percentage of that measurement. That can be really helpful to assess someone's immune function. Those are really the predominant reports we had, but we were able to add a lot of really interesting other reports.

I would say our aging reports really have two other reports. One is telomere length. And the other one is that rate of aging. And many of you might be familiar with telomere length. This is not measuring telomere length directly, but using methylation to estimate telomere length, which has some positives and negatives, but probably our favorite of all of the algorithms is the pace of aging metric. And that gives us an approximate idea of at this exact moment, what is your speedometer of aging? How many biological years are you aging right at this moment? And that's probably our favorite of all of the algorithms.

Dr. Jeffrey Gladden: We do get those four results. I think it is interesting to note that when Horvath was developing it, the closer they actually got to the actual chronological age the bigger the win was. Right? And of course that's, why would you pay for a test that's going to tell you how old you are? Because you know that, right? But I think moving away from the chronological clock, so to speak, to actually starting to look at these disease process clocks, and then also the rate of aging, it's really critical to understand what telomere lengths are. We know that telomeres in and of themselves are not the best biological clock. And there's some very clear reasons why that's the case. We won't go into those right now. But nonetheless telomere length is important.

It's also interesting to note that, if we can actually understand our rate of aging, it gives us some immediate feedback in terms of, what is our lifestyle choices? How is that affecting what we're doing? Am I exercising enough? Am I eating properly? Things like that. Let's just talk a little bit about the things that people can do that will impact their methylation. I was at a conference and was talking to people about changing their DNA methylation ages, and another scientist in the room raised their hand and said, well, you can't really change DNA methylation ages. I said, well, we see it all the time quite honestly. Actually we see it all the time with interventions, mostly around lifestyle choices. We see lots of changes.

And so do you want to talk a little bit about that in terms of how people actually impact this? What is accelerating it? What is decelerating it, if they're aging faster? And the clock that he's talking about here with your pace of aging, it's reported out as you're aging one year for each chronological year or you're aging 0.9 years or 0.8 years or 1.1 years or whatever it is. Do you want to talk a little bit about how people can impact that, what you've seen, what you've learned?

Ryan Smith: Definitely. And I usually split the interventions into two different types. One is the epidemiological interventions, and these are basically what we know from really large study cohorts, looking at associations, right? Correlations between behaviors and outcomes. And so that is probably the biggest body of evidence we have now, because a lot of those studies have been done, probably right around 150 studies have been done on these correlations to better or worse epigenetic aging. And generally the things that we see here are probably not the most exciting, the things that are relatively intuitive, right?

Things like maximizing your sleep, right? Getting seven hours of sleep, maybe not nine hours of sleep, right? Or having that special approximate of the best time, right? You don't want to underdo it, you don't want to overdo it. We see that type of optimal range for a lot of things, where we see the same thing for stress. We see the same thing for pollution and other environmental behaviors. We see the same things as it relates to diet and overfeeding. And so a lot of the things that we see epidemiologically are the things you probably already know from a behavioral standpoint that you should be doing.

I think where this gets really exciting is what we are doing interventionally. And those are the studies which are ongoing now. And there have been about 10 of those interventional studies published, where we're taking a baseline measurement, doing some type of protocol and then looking at an outcome measurement. And the first of those started really in 2019 with this TRIIM trial. And that was a baseline trial, which really set the precedence for being able to reverse or change these MET markers. They used the-

Dr. Jeffrey Gladden: This was Greg Fahy's trial. Greg's been on the podcast before and for the audience, this is the one where they were basically using growth hormone, metformin, DHEA, four days a week. And in a small group of men, I think nine men over a year and they showed a decrease in epigenetic age.

Ryan Smith: And so that one was the first piece of concept, obviously a little bit limited by its trial size with only nine patients. But now since then there have been other studies which have been published with several hundred patients, which look at some of these changes. And so there are some things we definitively know and now have been replicated in multiple trials. We know, for instance things like vitamin D now has three trials, which show that optimal vitamin D, especially if you're deficient can reverse your epigenetic age by several years, actually in these interventional trials. And so vitamin D is a big recommendation that we make. Going off of some of the work with Dr. Fahy, we looked at some of those constituents, growth hormone, metformin, and DHEA.

And surprisingly one of the biggest movers we saw for that entire cocktail was actually the DHEA. The DHEA seems to have a positive effect. We think probably due to its ability to mitigate the effects of cortisol and stress, which is a problem in the aging process and something we see high links to the level of self perceived stress definitely accelerates your epigenetic age. In addition to that, we're seeing things like diet nutrition regimens. We know that now the Mediterranean diet has three published studies showing that implementation of the Mediterranean diet can reverse epigenetic aging rates. We're starting to learn a lot of these things, but we're still absolutely at our infancy.

Dr. Jeffrey Gladden: Exactly. When you talk about the Mediterranean diet reversing epigenetic age, how many years are we talking about, over what period of time? Did somebody eat the Mediterranean diet for three months, a year or whatever, and how many years did they get back?

Ryan Smith: Generally there are a couple different studies on this, but the one that we reference the most often is a year long Mediterranean diet, interventional study. And the changes they saw in biological age were not massive. I think that that's an important thing to mention as well, is that it's not impossible to change these, but it is sometimes difficult. You really do need to change some of those lifestyles and you need to give it some time often. And so in that trial they weren't seeing massive age reductions, but they were seeing, anywhere from 0.6 to 0.8 year reductions over the course of a year, which is relatively significant, especially when you consider that they're still aging throughout that year.

I always want to temper expectations. That seven year age mark that we talk about sometimes can be very difficult to reach because you do need to do these things and implement these programs as a lifestyle and for relatively long periods of time. But there are other things I would say in the pipeline, which are very exciting that we're starting to see some really, I would say, big deltas for age reversal. And those are the things that I think we're really trying to focus on to have the biggest population of health impacts.

Dr. Jeffrey Gladden: There's a couple of things about that. There have been, we've been involved with some very small embryonic stem cells, and I know that you have been working with Dr. Todd on some of that as well. Right? And so what we've seen in using the very small embryo-like stem cells, and this is a particular form of stem cell that's endogenous to each of us, right? We each basically have a reserve of these embryonic stem cells in our system. And the reason that they go undetected is because they're attached to a protein, they're locked down by a protein if you will, in the blood. And they're very, very small. The whole cell is just about the size of a nucleus. Think of it as a dormant, hibernating lockdown stem cell in your system.

What Todd figured out how to do was actually to use laser energy to actually liberate those stem cells and activate them if you will. And so we all have these truly pluripotent stem cells in our system. I know from the conference calls that we have with him, that he's recording the data on people and I've even done it myself where epigenetic age has moved back for each treatment about three years and sometimes even four. I know we have some people that have moved back in time about 12 years epigenetic age and that sort of thing. Just to let the audience know, there are trials going on and we're just actually speaking with the IRB tomorrow night to get ours approved, which will include very small embryonic leg stem cells and DNA methylation age testing. Right? We're excited about that too.

But I think the other thing is that, I know you've been talking with some of the young plasma people also, and looking at some issues there, right? This is a little bit of the whole out of the parabiosis literature, where an old mouse makes a young mouse old and a young mouse makes an old mouse young. And the idea of removing things using plasmapheresis is one approach. And then the other is maybe to add back young plasma, which is the second stage of that approach, if you will. Or some people are just adding young plasma. Do you want to talk about any of that, what you've seen with any of those approaches?

Ryan Smith: I'd be happy to. I think that I want to caution just first and foremost that these are still relatively small powered studies.

Dr. Jeffrey Gladden: Very small.

Ryan Smith: So the trends that we're seeing here-

Dr. Jeffrey Gladden: Very small. Very small. Yes. This is bleeding edge right here. So just, right.

Ryan Smith: Definitely. But still give us some room for excitement, I think for sure. We first started doing some work on plasmapheresis and looking at some of the changes that we're seeing there. Plasmapheresis is essentially, in lay mans terms, very probably too simplistic lay mans terms of filtering of that plasma, where you're essentially taking out any factors which might be negative. That idea, again, I think started with the experiments that Dr. Gladden just mentioned, where they saw that when the plasma from old mice was given to young mice, they tended to have aging effects. So they accelerated prematurely, meaning that there might be something in that mixture, which is causing accelerated aging.

And then vice versa, whenever the old mouse was getting younger, the idea was that there's something in that young plasma which might be making that older mouse a little bit younger. And so we wanted to explore both, both just the filtering of older plasma, as well as the infusions of this younger plasma. The younger plasma so far looks very exciting. That's probably a little bit expected. They first did some of these young plasma fractions experiments in mice, and they saw upwards of 50% reductions in some of these epigenetic clocks, which was really, really exciting. And so it gave us a reason to believe that this might be an exciting therapy.

I think Dr. Horvath has even gone on record as saying it was a really particularly exciting mechanism to reverse this epigenetic age. And so in the trials we've done thus far, we actually saw something very interesting when we first started, which is that we actually saw these epigenic ages getting worse in the first two weeks. We actually saw multiyear age-

Dr. Jeffrey Gladden: This is fascinating. Right? I'm aware of this as well. People that got young plasma initially they aged in the first few weeks if they retested. Right? I don't want to steal your thunder, but go ahead.

Ryan Smith: No, no, you're absolutely right. I don't think that that was our anticipation. And so whenever we first got these, I think on our side, we were like, did we switch these samples? Is something happening here? But we repeated this and saw the exact same thing, which is a relatively significant but still relatively small increase in those epigenetic ages right around two weeks after those infusions. But then I think thereafter we've taken now trials of four weeks and six weeks. And we see that not only does that come back down to the baseline, but it goes beyond that.

And even significantly beyond that, and that's worth all of these clocks, the first generation, the second generation, and these third generation clocks like the DunedinPACE, we're seeing improvements in all of them, which gives us I think a lot of hope that maybe some of these therapies might be one of the ways to reverse the clocks. But again, I think that's another big thing to dimension there as well, is that these clocks haven't been around long enough to show that reverses of these clocks directly relate to improvements in these health factors. I think that it definitely suggests that, but we don't want to, I think claim too much.

And so we do think that people who reverse their age will absolutely help their health outcomes. I think we see this on these meta-analysis, these large scales, but we need trials like this that have multi-year age reversals to see how they're affecting the phenotypes. And that's why this is so exciting for us, is because with even just one therapy with plasma exchange, young plasma and plasmapheresis, we're seeing really big reversals in this marker, which gives us a lot of reason to be hopeful.

Dr. Jeffrey Gladden: Exactly. That's part of our IRB approved trial also. As you know, we're syncing a lot of things together actually to see if we can really go back. That being said, I think it is exciting. I think it's also important for the audience to understand that we're working in a space here where we're not quite sure what's cause and what's effect. In other words, we may be changing a marker, but are we really changing the underlying physiology? And so we're also in our trial, LIFE-RAFT trial, looking at elements of that too. And you and I have had some conversations about the transcriptomics and proteomics, and I know that you're involved with that as well.

Anyway, just so the audience understands that this is a very dynamic field and it's hard to look at one data point and say, well, that's the answer, because it has to be validated. And it has to be shown from looking at it from multiple different angles, not just through one angle, so to speak. But it is fascinating, because of all the things that we've ever worked with, we've never seen changes like this. And so that gives us cause for hope and cause for wanting to push on it further. Let's talk for a minute about this thing that's difficult to pronounce or certainly difficult to hear and understand what's been said, which is DunedinPACE. It's D-U-N-E-I-N-P-A-C-E, DunedinPACE.

When you hear, when you somebody rattles off DunedinPACE, it's like, what did they say? But it's DunedinPACE. I don't even know where that name came from exactly, but I know it was related to some people at Duke. And this is their rate of aging. Do you want to talk a little bit about some of the impact of the VSELs and the young plasma on the rate of aging also, and maybe break it down into the immune system and generally? We've talked in general about it moving back, but is it more selectively pushing back on the rate of aging or on the immune system or is there things that you've been seeing?

Ryan Smith: To give a little of background on that algorithm, again, it's Dunedin or duh-NEE-din, I think as it's pronounced in New Zealand. It's Actually named for the town in university, University of Otago, where this study occurred. This is the first ever third generation algorithm. It really has been done by studying the same individuals over the course of their life and looking at what epigenetic locations are changing in the same individual. So those longitudinal studies on epigenetic methylation don't happen very often. And so that's why this algorithm is one of our favorites.

It's also one of our favorites because we know that it responds to treatments which have already been established as really well validated mechanisms to reverse aging. In particular things like caloric restriction. The second and third generation clocks of GrimAge and Dunedin pace, both of those showed reductions in a two year study with caloric restriction, which is exactly what we would expect. We would expect that caloric restriction being such a well validated longevity intervention, would also see decreases in these markers, which measure the type of aging process. And that's exactly what we saw. But I also want to mention it's not what we saw for some of the first generation clocks that we're chronologically trained.

In that same cohort, those actually went up. And so again, what we're starting to do is with these second and third generation clocks, really drill into those aging mechanisms rather than just getting correlations. And so our signals are getting a lot clearer. And so that clock is our favorite for many reasons, but that is one of the biggest ones. This is one that we would oftentimes prioritize as one of the most important metrics that we provide as an output of a report. And so with the DunedinPACE, we see it definitely responds to things that maybe some things like the intrinsic or extrinsic ages don't.

And so in the case of both plasma exchange, or I should say in the case of young plasma transfers, we see that everything responds, every clock goes down and every clock goes down in a significant fashion, which is great. But oftentimes we might see discrepancies there. For instance, we did a senolytic trial where we used Dasatinib and Quercetin to clear out senescent cells and we didn't see movement in some of those other clocks, but we did see it with that rate of aging clock, which gave us, again, some reason I think to be hopeful that we were capturing maybe some markers of senescence in those clocks, which we know that those first generation clocks don't necessarily capture senescence, which is I think, why it's so important in for instance things like the LIFE-RAFT trial that you're getting those transcriptomic biomarkers as well.

We're starting to see that clock generally behaves in the way that we would expect it to, for the things that we know work. And that not only includes things like caloric restriction, but also things like these epidemiological factors, right? Stress and societal economic differences and things of that nature. And then we know that it's also correlated to quality of life metrics. Things like your facial appearance, your mental processing speeds and your IQ, and even your ability to balance or your muscle mass. And so we know it's not just about living longer, avoiding disease, it's also about improving quality of life.

And so that's why it's one of our favorite metrics. We're starting to see that changes in different forms and fashions to some of the other clocks, which gives us information about what's actually happening. And so some of the things we see there are even things like detox programs, getting rid of things, helping improving your ability for your liver to excrete different types of toxins seems to affect that metric pretty well. We're seeing, again, caloric restriction and things like mTOR inhibition through things rapid rapamycin, which can affect that metric.

And so we're really excited about that metric, but it does behave differently than some of the others and gives us definitely different insights.

Dr. Jeffrey Gladden: Yeah, no, that's fascinating. I think we've talked about LIFE-RAFT. It includes things like rapamycin analytics, plasmapheresis, young plasma, VSELs, and it's all being played together in this symphonic way where there is a timing frequency, intensity duration. The other thing about all of this for the audience to understand is we don't know how long these effects will last. Right? If you are able to turn back the clock, and I think it is nice that it's validating a lot of functional medicine, things like detox and eating well and sleeping, all those things get validated here, that they do not only improve your health and your performance and here and now, but you actually are improving your longevity in a way.

But we don't really know with these interventions what the duration of effect is. We don't know how frequently you might need to do young plasma, let's say, or plasmapheresis, or take a catalytic or use rapamycin or any of these things. We don't really have that dialed in yet, so we're trying to learn that as we go forward.

Ryan Smith: Exactly right. I think that's an area where we have a little bit of an advantage in the fact that a lot of the patients that we're working with are longitudinal patients. And as I mentioned, these longitudinal data sets they're very hard to find in this field. Most people don't have them. We now have over 1700 patients who have retested on our platform, which is great because we're able to follow their trajectories and see what their interventions are working and how long maybe some of those interventions are working. And so we're getting a bit of data to try and answer that question. What are intermediate benefits and what are lasting benefits?

I always like to put this in context with the paper that is just been released as a pre-print very recently from a group at Harvard where it looks at transient stress, things like pregnancy or things like surgery. What we see is that in those processes we see really rapid increases of epigenetic age, but only for a short period of time, generally right after the event they tend to go back down. And so that's the type of information we really need in order to [inaudible 00:36:16] what's lasting and what's not.

Dr. Jeffrey Gladden: That is fascinating. I've seen that data too, where if you go through a stressful time you do get older during that time, but your biology tends to pull you back to a younger age. It's not like it's a reset to that older age necessarily. So then the question becomes, if we push the system to a younger age, does it spring back? Does it rebound back to its more of its fundamental biological age, if you will, or starting point? Or are we actually able to push it back and that resets the clock? I think what we're all looking for is to reset the clock, not just manipulate the clock. I think that's really the big question that's out there, is what are we able to do to actually do that?

Ryan Smith: I think going in that direction it's impossible not to mention, I think cellular reprogramming, some of the things that we're seeing with these really big companies. I always like to mention one called Altos Labs, which was funded by Yuri Milner and Jeff Bezos. They just funded this company with three billion, all with this idea that they want to take these things called Yamanaka factors to reset these clocks back to zero, to take them back from this differentiated cell, all the way to a pluripotent stem cell, or to use some partial programming to really reset the clock, and then therefore have some functional differences.

And this is a really exciting, probably the most exciting area of age reversal, because we already know that in some animal species it's been able to do things like restoring vision from blind mice or to recreate some of the youthful skin appearance that we have with aging. And so it's a really big area of focus. One that is still probably very far away from clinical application-

Dr. Jeffrey Gladden: Agreed.

Ryan Smith: ... but at the same time one of the most exciting.

Dr. Jeffrey Gladden: No, I think that's right. Everything that we're doing and the things that you've been talking about are clinically available. They may need to be done inside of an IRB approved protocol, but they are clinically available. Using the economic factors to basically reprogram all your cells. That's going to be a week or two out I think before that goes live.

Ryan Smith: One of the biggest fears of that is obviously cancer risk. These growth factors have been shown to perhaps cancer risk. And so it is a long way away from actually being implemented, but the studies are ongoing and it's another area of hope I should say.

Dr. Jeffrey Gladden: Yeah, that's right. There were four of the economic factors if I'm not mistaken. It was when they used all four that they kept getting cancer, but when they dropped one of them and went to three or somehow mitigated the fourth one, I believe they stopped getting cancer. I know that there's some hope that they'll be able to do that and not precipitate malignancy, but to your point it's still a work in progress. I've got a question for you here. There are a lot of different companies out there doing epigenetic age testing. Is there any way that people can reasonably compare between the companies and know how to select a quality test or that kind of thing, do you have any insight you can shed for people?