The Peter Attia Drive - #118 - Lloyd Klickstein, M.D., Ph.D.: Rapamycin, mTOR inhibition, and the biology of aging

Episode Date: July 6, 2020

Lloyd Klickstein is the Chief Science Officer at resTORbio, a biopharmaceutical company that develops medications to target the biology of aging. In this episode, Lloyd discusses his company’s clinical application of rapamycin and its derivatives. He also elucidates details of his 2014 paper—a paper that greatly influenced Peter’s perspective of rapamycin in the context of longevity. Peter and Lloyd go on to discuss the dose-dependent effect of rapamycin on immune function and compare rapamycin, fasting, and caloric restriction.   We discuss: His background and decision to leave academia for translational medicine [6:15]; Translational medicine—bridging the gap between basic science and clinical medicine [10:30]; What prompted Lloyd to focus on mTOR inhibition? [18:00]; Defining mTOR, TORC1, and TORC2, and the consequences of inhibiting them with rapamycin [21:30]; Dose-dependent impact of rapamycin on immune function, mTOR inhibition, and toxicity [42:15]; Lloyd’s 2014 experiment—mTOR inhibition improves immune function in the elderly [53:00]; Insights into autophagy, antigen presentation, and the pleiotropic benefits of a rapalog, and how it compares to fasting [1:13:00]; Lloyd’s 2018 experiment—TORC1 inhibition enhances immune function and reduces infections in the elderly [1:18:45]; Creation of resTORbio, subsequent studies, and takeaways about dosing, TORC2 inhibition, and tissue selectivity [1:29:00]; Comparing the longevity effect of rapamycin, fasting, and caloric restriction [1:40:00]; Excitement around RTB101—resTORbio’s mTOR inhibiting molecule [1:47:00]; Identifying rapalogs selective for TORC1 [1:56:15]; Treating depression with ketamine, an activator of mTOR [2:00:00]; Epigenetic clocks, rapalogs, and metformin [2:03:30]; and More. Learn more: https://peterattiamd.com/ Show notes page for this episode: https://peterattiamd.com/LloydKlickstein Subscribe to receive exclusive subscriber-only content: https://peterattiamd.com/subscribe/ Sign up to receive Peter's email newsletter: https://peterattiamd.com/newsletter/ Connect with Peter on Facebook | Twitter | Instagram.

Hey everyone, welcome to the Drive Podcast. I'm your host, Peter Atia. This podcast, my website, and my weekly newsletter, I'll focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in-depth content if you want to take your knowledge of this space to the next level. At the end of this episode, I'll explain what those benefits are, or if you want to learn

more now, head over to peteratia MD dot com forward slash subscribe. Now without further delay, here's today's episode. I guess this week is Lloyd Clixstein. Lloyd's the Chief Scientific Officer at Restore Pio. So that's little RES big TOR little BIO. Get it Tor TOR. Restore Bio is a clinical-stated bio-farm company that develops meds that are primarily aimed at targeting Tor, targeted rap and mice. And we'll talk a lot about that throughout this episode. So prior to joining Restore Bio, Lloyd was the global head of translational medicine for the new indication and discovery unit at Novartis.

And prior to that, he was an academic physician at the Brigham and Women's Hospital, which is one of the flagship programs at Harvard. Lloyd received his bachelor's from Tufts and an MD-PhD from Harvard. He's got more accolades than you could shake a stick at. So accolades aside, the reason I wanted to speak with Lloyd was because he is really one of the few people on this planet that has a really nuanced understanding of the clinical

application of rapamycin and rapologues, and we talk a lot about one of them in particular, called Everolomus. Lloyd was the senior author on a paper that I have spoken about many times on this podcast, which we'll go into in great detail here. December 2014 paper, John Manic was the lead author on that paper. And that was the study that was basically the turning point in my personal evolution or thinking when it came to the use of rapamycin for the purpose of longevity. Prior to that, there had been a lot of studies that had certainly suggested in animal models that rapamycin could be a true longevity agent, but it was the manic, clickstein paper of December 2014

that was the real turning point in my thinking. And that's really where we're going in this discussion, along with talking about all that's been done since then. It is important before we start this interview that I mentioned, of course, that Lloyd is an employee of RestorBio, RestorBio is a for-profit company that is working on M. Torrinhibition. So please caveat everything that we discuss through that lens.

Before this podcast begins, I want to note that we recorded this interview in September 2019. In the interview, we discussed an upcoming phase three trial from Restore Bio. Since that time, the results have been published, and the study did not meet its primary endpoint. Now, I frankly left the option to Lloyd, as to whether or not he wanted to still have the

podcast air and he felt that that would be fine to do. And so we're going to go ahead with it. And eventually I'm going to be interviewing his colleague, Joan Mannack, along with near Barzalheim and have the two of them back on an episode where we're going to discuss a whole bunch of things that will be quite interesting. And this gets more complicated because I think I have a pretty clear understanding of why that study failed and what it does and doesn't say about selective inhibition of agents like it.

Nevertheless, I think the most logical thing to do is to go ahead and proceed with this interview, which is one of my favorite interviews on this subject matter. And just know that there are going to be a number of things that are left as open-ended questions from this discussion. We're going to pick back up with Joan Manic when we do that interview, which I'm scheduled to do a few weeks from now. And hopefully we'll try to get that one out at a much quicker turnaround. There are a number of issues that delayed the release of this, not the least of which being some of the COVID stuff,

but I can promise you that there will be a shorter gap between when you are hearing this and you will hear the follow up to this than there was between the recording of this and when you're hearing it. And without further delay, please enjoy my conversation with Lloyd Clixty. Oh!

Oh! Oh! Oh! Oh! Oh! Oh! Lloyd, thank you so much for making the trip up to San Francisco today. I know you didn't come here specifically to see me, but I appreciate you carving out a little extra time to meet today. I'm happy to be here and looking forward to our talk.

I hope the view is enticing enough here. It's lovely. When the weather is nice in San Francisco, there are a few things that compare to it. And when it's not, it's like Mark Twain said, right? The worst winter he ever had was a summer in San Francisco or something to learn about. And here from Boston, so you laugh at that. Right now, this is the weather we all wish we had in Boston.

Yeah. So Tim Wright, one of your colleagues, offered to make this introduction over dinner one night. And there's probably never been in the history of an introduction from the moment the intro was offered until I was sitting down talking to someone on a podcast that was quicker than this one. In other words, I'm sorry to hear that actually. Well, it was just a meaning I was so excited when we sat there and it was so as with Tim

and with DA, who some folks listening will know because I've interviewed DA on the podcast as well, and we were having this dinner. And as is always the case when I'm talking with Dorky Science friends, Rapamycin comes up, and one thing led to another, and then I'm embarrassed to say this, but the 2014 paper that I talk about constantly,

I was referred to as the manic paper, because she's of course the first author, but you're sort of the lead author, you're the final author, you're the senior author on that paper. And so I was embarrassed to say this, I didn't even, when they mentioned your name, I didn't put two and two together. And I didn't know you were at restore bio at the time either. So anyway, they connected us, we communicated over email, the rest is history we're sitting here today and I am beyond excited.

And then this is a topic that I just know listeners are dying to hear about because it's been over a year since I've had a podcast on this topic. So very early in this podcast, which is about a year and a half ago, we had discussions with David Sabatini and with Matt Kaverlin, who are both amazing folks and legends in this area as well. So I'm going to discipline myself for a moment before getting right into the rapist stuff to give a little bit of background. You've got a pretty interesting background. I want to hear a little bit about it. When did you realize this is what you wanted to do, which was basically physician scientist, and then ultimately now moving to industry?

Well, I guess I can begin by stating that science and medicine is the family business. So it wasn't much of a stretch for me to be here, doing what I'm doing now, doing what I've done before, on both sides of my family, both sides of my kids' family, all of my kids are scientists, doctors are both. So did you do a combined MDPH day or did you do them separately? I did the combined one. My wife did them separately, actually.

The long and expensive way. Yeah, exactly. At least when you do them together, they pay for each other. Yeah. When did you decide you wanted to focus on immunology, rheumatology? I mean, there's no shortage of things one can specialize in. One of, at least my challenge is that I know the challenge

of many physicians and many scientists is that so many things are interesting, how do you focus? And like many things in life it was about the people, not the science that led me into immunology, rheumatology, and where I am. When I had left college and wasn't sure where I was going to go next, I spent a couple of years working in the laboratory at Brigham and Women's Hospital. I had such an incredible time and met such wonderful people that ultimately my decision was to stay there

and work with them, learn from them. What years were you at the Brigham? I worked in a technical capacity from 79 to 81. Started medical school in 81 and finished both degrees by 1989 and then stayed there through all of my training and then left in 2006 to join, actually, Tim Wright's department at Novartis Institutes. What prompted that decision? And is that a one-way street for most people? There are people who go back and forth, but I think we have to be realistic that it's more challenging to go back to academia if you don't have extant grants and external funding.

It has to come from somewhere in most places. In terms of what drove the decision, I'm a physician scientist. For me, it's important to do both. I'm a physician scientist. For me, it's important to do both. Science and medicine. And it's harder and harder and harder to do that now in an academic environment, at least. I'll get in trouble for saying this, but at least a primitive academic environment like Harvard, where you eat what you kill and you have to,

at the same time, see patients, be at the top of not just your game, but the world's game in seeing patients and administrative and teaching responsibilities and so forth. Well, I mean, let Harvard get upset at you for saying that, but I mean, there's no denying what you're saying is the case. Every, I interview so many people who are straddling that in, I'm constantly amazed. In fact, I was interviewing someone recently a very remarkable scientist and academic,

and I couldn't believe how much clinical obligation he had and yet how prolific he was. It's sort of amazing to me that some folks can actually straddle that it's certainly not optimal, I guess, as the point. No, and it was much more challenging than I had seen in the late 70s and early 80s. What changed the reduction in grant? The competitiveness of the grant environment? No, it wasn't

so much the grant environment. It was more the regulation and the paperwork that was imposed mostly on the clinical side. I need to be fair and say I was both running from something and running to something. As a physician scientist, the goal is to have each of them contribute to the other and translational medicine, which was a new concept around the turn of the millennium, was growing and was perfect for somebody like me. The Vardus Institute was created by Mark Fischman in the early 2000s as a new concept and a new approach to drug development, thinking about pathway biology, and they were building translational medicine departments, and Tim recruited me to lead the musculoskeletal one.

So maybe explain to folks the difference between basic science, clinical science, or clinical research, and translational research, which as you said, the latter there being a relatively recent phenomenon. Lots of examples of basic science. One that's pretty exciting and as led to Nobel Prizes is the study of restriction enzymes. Who thought that studying obscure bacteria and how they limit their infection by viruses might have led to the concept of restriction enzymes, which was required for the development of modern molecular biology? Another one of basic science, you've probably talked about CRISPR technology here. We haven't had dedicated podcasts to it and I love to get Jennifer on to do so. But please continue, yeah, that's a great example. I'll give you a one-minute

summary. It is another critical element of the bacterial immune system. It's simple, it's elegant, it's powerful, and there were scientists in Europe studying fermentation for yogurt and cheese, and they discovered CRISPR-Genefer-Daudna and colleagues here, MIT, and the Broad Institute, and they were studying basic science. They were studying bacterial biology, and it became so exciting when somebody translated the biochemistry, if you will, in the bacteria to see what it would work in humans. Who would have thought that would work? Bacterial chromatin, the DNA is so different, it's supercoiled in a bacterial cell, where as human DNAs organized into chromatin and methylated, but it did.

So the point here is, basic science isn't necessarily in pursuit of anything beyond knowledge, but it doesn't come with the caveat of this needs to have a clinical application with respect to the species of interest. Exactly right. Clinical medicine, I think everybody knows. It's getting your flu shot in the fall. It's being told to diet and exercise and take your anti-hypertensis if they've been prescribed.

Right. Does taking this medication lower your risk of a stroke does taking this vaccine lower your risk of getting the flu. Exactly. And translational medicine, there's a big gap between those two, isn't there? Yes. And that's what translational medicine does. People had been doing this for a long time, but never in an organized and conceptually

holistic way, if I could say that. It's how do you take a basic science observation and make something useful for human health out of it and prove it, which is surprisingly challenging. So you're saying that basically up until roughly 20 years ago, this hasn't been particularly well organized. And now, pharma companies, among others, are saying we'd like to own some of this risk. And I think everybody has bought into the concept of translational medicine now,

probably all big pharma companies and many small ones do it. Many academic institutions have organizations to do translational medicine. In part, our responsibility, it's in part a way to do it more efficiently by providing appropriate training and experience to younger scientists. We always have to think back to who's funding our basic science, especially in academia. Ultimately, it's the people.

Most heavily taxpayer funded. Yes, it's Most heavily taxpayer funded. Yes, it's most heavily taxpayer funded and the reason they're doing it is to make their lives better or the lives of their family and friends better and we need to be better at that and I think this is taking a step in that direction. So what was the first translational problem then you began to work on when you joined a partisan the what would have been I guess the 90s, right? It was in the early 2000s.

So I joined a musculoskeletal program at the time of artists was working on, it was a regenerative medicine concept and musculoskeletal biology to increase bone density and increase muscle strength. And so we had to put together some programs that would translate some basic biology observations from human genetics. Remember translation can go both ways. And in fact, to skip ahead a little bit, we actually did that from that 2014 paper. We took what we learned there and went back into the mouse. And there were a few projects. The one that has been successful is drug called

Zolodronic Acid to increase bond density. And what was the state of assets that you came into? Did Novartis already have a basic program that had shown some new insight with respect to biology that could then be extrapolated into a compound. Did they already have the compound? What was the actual program you were creating at that time? Novartis worked very much like most other pharmaceutical companies did. There was a basic science department led by PhD scientists. there was a clinical development organization

led by clinical developers, including MDs. And there was a throw it over the wall mentality. The scientists make something that they like, and they throw it over to the clinical scientists, and they have to figure out what to do with it. This is pre-IND? Yes. Okay. We should explain what an IND, and I suppose it's a...

Yeah, do you want to I&D and I suppose is yeah, do you know folks about that? Sure, so an I&D is stands for Investigational New Drug and this is the application that sponsors make to the FDA to get permission to begin clinical trials and there are a whole raft of requirements that are necessary in terms of quality, manufacturing, clinical plans, risks and benefits, experiments, and so forth so that we can make it as safe as possible to test something new in people. But in any event, that's the way Novartis worked as they separated research from clinical development. Other companies do it a little differently.

They had the initial testing in healthy volunteers or the initial testing in patients depending on the risk benefit argument as part of the research organization. But in principle, it's the same thing. You had scientists making medicines and then clinicians testing them. The goal of translational medicine is to provide clinical input right at the very beginning of the process, even when you're thinking about what to do. And it really helps to focus the drug development process on the patients and the clinical need, right from the beginning, so that ultimately the drug that's made is the drug that's needed. Not let's make something and figure out what we can do with it. Let's figure out

what we need and then make it. Is there evidence by the way this is a bit of a tangent, but that that transition has rendered pharma more efficient at yielding capital. This is studied in an anonymized fashion by an industry organization. it varies by company. I see. So let's fast forward a little bit to the first time you became involved in a molecule that would be involved in a nutrient sensing pathway. What was your foray into that? There's a step before that, and that is how did I get from musculoskeletal disease to something new. We have to credit, again, Mark Fischman, who is the founder of the Novartis Institutes

for this, because he challenged me and a few others to put together an organization within the company and answer the question, what aren't we doing that we should be doing? And then start some projects. And again, it's all about medical need and of course scientific tractability. So we started that project. We called it the New Indication Discovery Unit. What year is this roughly mid-2000? This would have been about 2008 or 2009. We basically applied those principles, a real medical need, a problem that's scientifically tractable, and that Novartis wasn't working on an ideally that nobody was working on.

And we ended up with a few very interesting areas. Joan Manek, who was the first author on that 2014 paper, brought the idea forward. Well, maybe the biology of aging is tractable, but how do we actually make a medicine and develop it? That was Joan Atnovartis at this time? Yes, she joined our group in part to do this. Her real innovation, beyond just the ideas that she figured out a way to test a medicine

that could alter the biology of aging in humans and find an endpoint that's measurable and modifiable in a reasonable time frame. Which really is the Achilles heel of aging research, which is the ultimate outcome is virtually unmeasurable in the species of interest. Yes. So our approach is not necessarily what you might read in the popular press about making medicines for aging. Our approach is to address serious, aging associated diseases. And if we're successful, the side effect will be longevity.

Yeah. So keep going then. Now, Joan floats this idea, which is, here's a really good proxy for aging that can be measured out in a time course that's clinically tractable and also, frankly, amenable to the type of research that we can do in humans. And so, what was your aha moment? This is interesting. This is interesting to Lloyd. I needed and beyond interesting to Lloyd, we then as a team had to persuade the rest of the organization, hey, let's try this idea.

And again, we always come back to the medical need, scientific tractability, and in proposing a project, what's the evidence that it's going to be successful? And you know as well as anybody, there's substantial scientific data that MTOR inhibition will extend health span in many preclinical species, certainly all the ones that have been tested. Now, that was not obvious in 2008.

I mean, 2009, people had been speculating, and of course, there was a major publication that came out in 2009. I have to correct the timeframe. We started our new indication discovery unit in 2008 or 2009. I think Jones started the project in 2010. Got it. So you already had a very important study behind you as a catalyst for that. Let's take a step back now and explain because

it's been a while and there's going to be people listening who don't recall all the details of our discussion with David Sabatini and with Matt Kaibberlin. Let's talk about what is MTOR? Sure. Well, I can't add anything to David Sabatini about what MTOR is. Nobody can. But let's assume people have not heard what David has to say. Sure. In a nutshell, MTOR is the master integrator of external availability of nutrients and growth factors, and then is the master regulator of the outputs of that integration, deciding whether cells are going to make proteins, make lipids, make nucleic acids grow, or are we going to circle the wagons, conserve resources, recycle, and wait during times of little for hopefully future times of plenty.

So that's the role of MTOR. So it takes a bunch of signals, which are external to the cell, ultimately become internal to the cell, because mTOR is in the cell, not out of the cell. It assimilates and integrates across that signal and makes decisions that lead to, as you said, there is go over simplifying, grow or don't grow. Yes, I think that's exactly right.

The signals that takes are amino acids, glucose, cellular energy, growth factors from other parts of the organism, as probably the major ones, and then decides, are there sufficient resources that the cells should grow or not? And between, this is just a little history lesson for the listener. So, between 1991, when Hall first identified what was not called at the time tour, but what would go on to become tour in yeast and 94 when Sabatini identifies it in mammals, you basically had some of the just the heaviest hitters in biology, all sort of converging on this idea, which is this is a really ubiquitous thing that has been preserved

across about a billion years of evolution with very little change. You don't see that every day in biology. Why is that relevant? The simplest argument is that things that have been concerned from single cell organisms to us are probably important. There's some interesting comparative zoology that's relevant to mTOR here. If you think about where in the cell mTOR lives, it's active on something called a lysis

sum. And that is a structure in the cells that's responsible for breaking down either cellular material or material that's been acquired from outside the cell into its component elements that then could be recycled like amino acids and sugars and so forth. Very early in development, well, in evolutionary biology when there were single-celled organisms and then the early multiple cellular organisms. The way that the organism ate was by creating a vacuole from whatever was on the outside and then creating a lysosome. So we'll consider pictures endocytotic process is the cell membrane or wall depending on what if it's eukaryotic or pro-cariotic

sort of sucks in a little bit, which creates basically a space, and then the outer parts of that wall reach up, reach around it, and can actually seal, and now you've created like a vacuel that you pull into the cell. Yes, and in the early multicellular organisms, there were specialized cells for doing this and they were called phagocytes for eating cells. Later on, it was learned that phagocytes could also serve an immunologic role, in other words that they could eat pathogens as well as nutrients. This happened in the late 1800s when higher quality microscopes were available. A Russian scientist named Ilya Mechnekov did a lot of the pioneering work on this. He was working in Paris. And he described, he was an embryologist and comparative zoologist. And he described by looking at small animals that were completely transparent so he

could see all the cells inside and what they were doing. He actually imaged them while they were alive and he could watch them eat and he could see all the cells inside and what they were doing. He actually imaged them while they were alive and he could watch them eat and he could watch them fight bacterial infections. And he was the major champion of something called cellular immunity. At the same time, some German scientists notably Paul Erlich were working on what we now understand as antibodies.

And they said, no, it's humoral immunity or soluble immunity in your blood, and they had the cellular immunologists in Paris, and we had the humoral immunologists in Germany. Eventually, they figured out they were both right, and they both got the Nobel Prize in 1908. But this is why M-tours, probably on a lysosomal vacuole because in the context of evolutionary development, it was on these vacuoles that very simplest organisms used to ingest food and nutrients.

And so you want to have it close to, because it's there to sense those things, you want to have it very close to where it's there to sense those things, you want to have it very close to where they enter the cell. Yes, exactly. So if we take a given eukaryotic cell today, take one of our cells, how many mTOR complexes would exist in a cell? What order of magnitude?

I turf that question to David Sabatini. But I don't think I've ever asked David that question. I don't know. I would guess it would be on the order of thousands, not millions, not tens. So one of the other things that David's done is not just recognizing this in mammals, but also recognizing that M-Tor, which again, it's one of those things that's funny when you start to explain it to people because you can can't explain what MTOR is without somewhere explaining what RAPA MISON is given the name. MTOR stands for Mechanistic Target of RAPA MISON.

But David also played the fundamental role in elucidating that MTOR can be organized in a couple of different ways, in two main different ways it can be organized, known as complex one and complex two. Explain a little bit about what those two mean. How do they organize differently? And perhaps more importantly, is there a functional difference between those? Sure. So in yeast, there are two separate tour proteins, one and two. And in, I think all other species, there's just one mTOR protein, and it can be assembled into two different complexes. One of them, or called torque one, for targeted rapamycin complex one, regulates many of the

things we've been talking about. So protein synthesis, lipid biosynthesis, protein translation, and so forth. The other complex, torque 2, regulates cytoskeletal, so in other words, the skeleton of the cells organization and growth decisions. So different. Now this is sort of interesting, so let's talk about rapamycin now. How does rapamycin interact with TOR? It's target. That's an excellent question because you think about TOR being the target of rapamycin. It's not exactly the target of rapamycin

is an immunofillin called FK binding proteins or FKBP. And there's several of these. There's three different classes of immunophilance. The complex of rapamycin bound to FKBP then binds to the torque 1 complex and inhibits it. And it inhibits it in different ways for different downstream targets. The one that's most commonly measured is something called phosphosix kinase, which names not important, it's just this is the protein translation pathway, and it's very efficient at inhibiting that. A little less so for another target called 4 EVP1, and even less so for a target called ULK1, which is involved in activating the cells

recycling machinery called autophagy. In other words, let's go through that again. So rapamycin binds, and how tightly does it bind, by the way? Pretty tightly. So, binds pretty tightly to this binding protein. This binding protein then moves towards tour. And in the case of, did we explain raptor and Richter yet? We haven't explained those case. Do you wanna spend maybe just a minute

so that they can see the difference between complex one and complex two? So M-tour is present in both torque one and torque two complex. But there are proteins that are unique to each complex. So, as you were saying, the yeast have two different tour. Everything else has the same tour, but it's another binding protein. It's another protein bound to it that creates the distinction between complex one and two, correct? Exactly.

And we should qualify every organism we've looked at as only one m-tore. I'm sure we haven't looked at all of them. So, Raptor and Richter, again, discovered in David Sabatini's group, Raptor is unique to Torque One Complex and Richter is unique to the Torque Two Complex. And they're covalently bound to Torre? I don't think it's covalent.

So it's some sort of conformational configuration, but not necessarily. Well, it's a multi-subunit complex, but I think they bind on the basis of having a fairly large surface of interaction, not covalent. And then the complex, to get back to the FKBP of FKBP plus rapamycin, then binds to the the torque one complex and inhibits it. But again, it inhibits it very well for some of the downstream pathways and not so well for some of the others. And let's review, again, those three. So the first one that binds really well, the serine phosphate is which one?

There's the binding interaction of the RAPA micein FKBP to torque one. Yes. And then that alP to torque 1. Yes. And then that alters the torque 1 downstream activity. It inhibits quite effectively phosphorylation of S6 kinase. S6 is a critical protein in the ribosome required for protein translation. It works a little less well for a protein called 4EBP1, which is an inhibitor of protein translation, so you inhibit the inhibitor and you activate protein translation. And it's less effective at phosphorylating ULK1, which is an early step in the activation

of the cell recycling machinery called autophagy. The interpretation of that is as following, RAPA-MISIN is a strong inhibitor of making new protein and a modest activator of autophagy. Exactly. Is that a fair statement? Yeah, I think that's a fair statement. We're talking about these pathways as specific examples. Remember, torque one does other things too,

particularly in terms of regulation of lipid synthesis, perimidine synthesis, perimidines are part of DNA. What about M-tork two? So how does RAPA and FKBP bind to M-tork complex two? I don't think it does directly because there's no immediate effect of the complex on torque 2 activity.

If you look at the downstream targets, they're not affected in the short term. There's a longer term feedback inhibition of torque 2. And is that more due to the failure to recent the size enough tour? Is there a shortfall of tour because so much of the tour is bound to the Rapa FKBP complex that you now run out of enough tour to make M-tour complex too? You could imagine that is one possibility, but I don't think that's the case, I think it's

more of a feedback signaling pathway that downregulates torque two. What you said is very important, and we're going to come back to it in great detail, but there's something temporal about this, isn't there? Yes. Do we know how much exposure to rapamycin or a rapologue is necessary, constitutive exposure, before you start to see this dual prong of inhibition? Are we talking about a day, two days, three days, a month? I know you had David Sinclair on recently. Yeah. And I listened to your talk

with him. He talks about mouse experiments. My bias is to talk about people experiments given my role in humans. After a week to a month, you can start to see consequences of torque two inhibition with a rapologue alone, and it's reflected in hyperglycemia and hypertocularceridemia. So biomarkers in the peripheral blood you can measure. Why is it that inhibition of m-tork two leads to that phenotype you just described? I don't think we know exactly. Is it confirmed that that phenotype you just described. I don't think we know exactly. Does it confirm that that phenotype exists in healthy volunteers?

In other words, we see this for sure in patients who take rapamycin or its analogs in the context of organ transplantation, but if we took non-diabetic, non-immuniconpromised, quote-unquote as normal as possible subjects, do we have evidence that those things happen? We do. MTOR inhibitors, well, rapolog specifically, have been tested in non-transplant, non-malignant

disease patients. Some specific examples include the rapolog rad 001 was tested in patients with polycystic kidney disease. These people are basically well except for that renal disease. And even in those patients there were a substantial fraction who saw these biochemical changes in their blood. Now not everybody gets it. We don't understand that either. Do we know what the dose equivalence was of rad001 versus rapamycin? In other words, they were getting it daily. Do you recall at what dose did you start to see this consequence?

That was a phase three study. It's published so you can look it up. If I recall the dose was 10 milligrams a day and I think they had an opportunity to decrease the dose to five milligrams if it wasn't tolerated. Is rad 001 identical to rapamycin endosing or what's the dose equivalence? No, again, it's hard to do an exact dose equivalence because the biochemistry of how exactly the complex works with the mTORC complex is a little different.

But if your repamycin dose is somewhere between two and eight milligrams a day roughly at the immunosuppression level of dosing which is what we're talking about that's comparable to five to ten milligrams of red zero zero one. Got it. So they're pretty similar but not identical. Yes. So let's put a bow on this particular question and then take a step backwards. Is it safe to say that most of the inhibition of mTOR complex to seems to produce things that are not really desirable at all? Whereas the output of an mTOR complex 1 inhibition pathway seems quite desirable at least some time. Yes, and in fact, most genetic experiments have supported that conclusion.

Let's talk a little bit about those experiments. So if you genetically knock out Raptor, so that you no longer have complex 1, but you still have mTOR andore and rictor, so therefore you have complex two. What does that animal look like? It depends on how you do it. If you imagine making a mouse that doesn't have raptor one, which means it doesn't have torque complex one, from conception, I assume it has muscular dystrophy or something like that, or... They're embryonic lethal. Yeah, okay. You need torque one for development. So if you just turn it down by some amount, 50% reduction, not much of a phenotype of that, but the way the experiments have been done is you can conditionally knock out a target

in a mouse experiment. So you can create an experiment where the mouse is normal and is born and develops normally. And then when the mouse is a young adult, you can knock out Raptor, knock out Torque One. So once they're out of the development window, then they've reached adult size. Then it's better tolerated. Yeah.

And again, David Sapatini has done a lot of these experiments. His group published a nice paper not that long ago where they knocked out several components of the Torque One complex. Inhibition of Torque One extends lifespan and health span and rodents. If you do that to Torque Two, it accelerates death. It's such an interesting concept.

I mean, because what it basically suggests is, at least with the tool that we currently have to block tour, which is like Rappamysen or Rappelog, giving intermittent dosing, maybe beneficial, giving constant dosing may cancel out the benefit. It's hard to know, because when you give it constantly, you're getting the quote-unquote good inhibition and the bad inhibition. You don't know what the net effect is, right? If you're using a rapologue with continuous administration, yes, you'll eventually downregulate torque too as well.

And as far as we know, that's not favorable. There are some other ways to do this. And Joan Manick's second paper from 2018 was the first time we've explored that in humans. I want to come back to the 2018 paper, but I want to build up to the 2014 paper. In 2009, this mouse study comes out. It was the first of what would become a series of very interesting, highly reproduced ITPs, funded by NIH, that sort of did something we didn't typically see,

which is consistently across multiple labs and across different strains show the same result. A lot of times in biology, you just don't get that. You get the one-hit wonder, and it doesn't work in any other model or in any other lab. And that's not because people were nefarious. It's just there's some very very particular niche sort of circumstances that are being exploited that we don't even understand. That didn't seem to be the case in Rapa Mison. On a personal level by 2009, I am now very interested in this compound.

But I don't know what to make of it because I remember being a resident at the hospital giving lots of serolumus, a rapamune, to transplant patients along with their prednisone and their other immunosuppressive drugs. And there didn't seem to be anything about that that seemed to be longevity producing. It didn't make sense that you could suppress the immune system and somehow reduce death. It seemed counterintuitive. I mean, in the transplant patient it made sense, because of course their greatest risk is by far organ rejection, but these animal studies were not replicating that. So, I remember being incredibly confused for about the next five years.

How did you guys start thinking about that problem inside Novartis? Joan proposed the idea, and again the real innovation was being able to recognize that I'm doing air quotes for the listener's age of the immune system is something that's measurable and potentially modifiable in a reasonable time frame. And we had that paper from Chen in Science Cell signaling that showed a short course of a rapologue could alter the biology of lymphocytes. And you're saying that in a favorable way, not a disfavorable way. Yes, yes.

Because the earliest observations of serencigal were that lymphocytes being highly proliferative were heavily targeted by rapamycin. It's all about the dose. Yeah. So let's talk about those doses. As you alluded to earlier, a transplant patient might be taking 5 milligrams a day of rapamycin, day in and day out.

What types of doses were you seeing that were producing this counterintuitive phenotype? Well, if we get back to the mouse papers... Yeah, yeah, exactly. Like, lower or higher, I guess is what I'm saying relative to that it's hard to compare is you have to look at exposures So the doses on a weight basis or a body so much higher tip or much higher in a rodent experiment But the exposures can be comparable they were actually fairly high in his paper They were at least equal to what we what we use in transplant high in his paper. There were at least equal to what we what we use in transplant. Obviously we couldn't do that in healthy volunteers, especially healthy elderly

volunteers. There was some additional information that gave us some confidence we could use much lower doses than what was used in transplant patients, yielding much lower exposures. And for the listener's exposures means how much of the drug is actually in your body over time. So let's use Cialis as an example of this. I don't know why, but I was talking to a patient about this the other day. Cialis is typically given as either 5 milligrams or 20 milligrams, and patients typically have a choice if they want to take 20 milligrams, quote unquote, on demand. So you're heading into the weekend, you're going away with your wife.

It's Friday. You take the 20 milligrams of Cialis and rectal dysfunction is ameliorated Friday through Sunday. Conversely, another way to take Cialis is to take 5 milligrams every single day, whether or not you're going to be sexually active or not, but now all of a sudden, anytime you want to be sexually active, you're functionally like that person who just took 20. Use the lingo of exposure to explain how those two things are comparable. I don't know why this is somehow the first example

that came to my mind, but probably just because it was a discussion I had two days ago. It's actually a good example because when you take the medicine, doesn't always reflect if the medicine is in your body or not, and medicines that work have to be in your body to work. So the 5 milligram dose of psialis, So the 5 milligram dose of psialis taken once might be in your body for a day. A 20 milligram dose of psialis taken once is in your body for two to three days. So it's why the higher dose taken once can last over the even long weekend perhaps, because it's still there. This we call in drug development,

a half-life of the drug. So the half-life of psialis is long enough that it can do that. There's not the case for Viagra, for example. That's right. Now, there's another interesting thing here, which will also be another, we'll have a parallel to the RAPA story, which is, generally, patients will tolerate 5 milligrams daily of chialis more than 20 milligrams on demand because of the side effects. which is generally patients will tolerate 5 milligrams daily of

Cialis more than 20 milligrams on demand because of the side effects. You have fewer side effects because you don't have the same peak levels. So 5 milligrams daily will produce a very consistent and narrow gap between peak and trough, which is therapeutic, whereas 20 will overshoot. You'll get a very high peak level, which may increase the side effects, lightheadedness, changes in vision, things like that. And then you have a long enough way down before you hit trough, and it's during that entire window that you have the availability of the effect of the drug.

Now, that's true for the class of drugs, the phosphid Astray's inhibitors, yeah. P5 inhibitors. It's not true for other drugs. Two specific examples, rapamycin, where if you remember when you were treating your transplant patients, you measured trough levels.

Yeah, daily, I mean, we were constantly doing this. But you measured the trough levels, not the peak levels. That's right, because the side effects are driven by the trough levels as well as the efficacy. And I think that's also true with gentomisin, and a lot of the antibiotics that have the same toxicities on the trough, right? Yes. So gentomisin years ago, we used to dose three times a day, and modern times we dose it once a day, and we get better efficacy and far fewer side effects. So why is that the case?

That a drug like gentimicin or rapamycin is producing toxicity by its native, not its peak? Every drug is different and it depends on the specific mechanism for gentim isin and amine glycosides in general. Remember, these work on the bacterial ribosome, and there's some congruency between mitochondrial ribosomes and bacterial ribosomes, and with sustained inhibition that can cause toxicity, particularly in the kidney and the tubular epithelial cells, also in

inner ear hair cells and some other places. So having some drug-free time seems to allow the organelles to recover, at least that's the hypothesis I heard in medical school. We all know that half of what we learned in medical school is wrong and they just weren't sure which half. You had a better medical school than me, I think 90% of what I learned in medical school is wrong But you went to Harvard. I only went to Stanford So I think that's the West Coast East Coast difference. No, I think that's I mean to me that is the most logical argument which is Drugs that have trough toxicity are drugs where you must have a break from the drug and the higher the trough the lower the probability of a break

and the higher the trough, the lower the probability of a break. Peak drugs aren't about time away. It's literally too much of this thing eventually hits a trigger. That's probably an oversimplification, but it's a useful conceptual framework. So now taking that model back, the first glimmer of hope that this drug had wasn't uniformly immune suppressing was, yeah, what if we dose this lower, basically? And not from the standpoint of side effects because that's a common reason you'd go lower,

but actually change the profile of inhibition. Was it known at the time that that's what they were trying to do, like did they have enough insight into how rapamycin bound to the two different complexes to test this hypothesis proactively, or was this more empirical and observation that after the fact the mechanism became elucidated? We had some information up front. One bit of information that the mechanism could be favorable for immune function, not solely

immunosuppressive, was looking again in your transplant patients at those who were on a calcinerin inhibitor versus those who were on a rapologue, and there was a significant trend that those on rapologs had fewer side of megalovirus infections than those on calcinerin inhibitors, all of the things being equal. An observational study, not as well controlled as ideally we would like, but it was intriguing. Do you remember the order of magnitude on that difference? So you're basically talking about FK 506 versus cyclosporin. Yeah.

Well, it was rapamycin versus cyclosporin or FK. Right. Maybe I don't remember we'd have to go back and look. So that was item one. Item two is we had done exposure response experiments in cellular systems, looking at how much drug is required to inhibit the downstream targets of TORQ1. And it was much, much lower than the exposures that are observed in the usual dosing framework of rapamycin, at least for transplant and immunosuppression. If we were going to be treating healthy people with a

rapologue and test whether their immune function was better, we couldn't be giving them a typical rapologue side effect. And this is, again, a critical element of the translational medicine that Joan did in order to make this proposal is what doses and what schedule would be required to keep the trough levels actually less than assay, which would be as safe as we could get it, plus nonetheless achieve adequate exposures to at least partially or temporarily fully inhibit torque one. That would let us ask the question. Let's now pause for a moment to explain. We've switched back and forth between the term rapa mysin and rapa log.

So again, a little bit of a history lesson, but rapa mysin is the name given by Sirren Segal to the compound identified on Easter Island that went through two companies before being eventually absorbed by Pfizer through Wyeth, and that was a drug named Rapamune or Sirolamus, and that was FDA approved in 1999 for transplantation. What was the first rapa log to come along. Arguably it's RAPA-MICEN. The next drug... Yeah, sorry, with the Atlantic's aside. Yeah, after RAPA-MUNE, RAPA-MICEN, SOROLAMAS, we are talking about the experiment that we're about to discuss in detail is using a different molecule. It's using a different molecule. We called it

red-001 in the paper. The generic name is Evarolimus. It was the second one, Tim Siralitis is another one. How does it differ from rap and muonor serolimus? There's small structural changes, and it was synthesized to be different versus discovered in nature or was it deliberately modified presumably? Yes. Again, to improve the properties of the compound. Has that borne out? I mean, obviously there's an IP reason one would do that, obviously, but in terms of clinical efficacy, are there differences? I'm not aware of any comparative studies. In vitro, potency seems to be a little greater.

I can tell you from firsthand experience looking at the cost of these drugs, there certainly is a difference. Good Lord. Yes, it's generic versus brand I think at this point still. But even Rapimune branded compared to, but anyway, I am not somebody who can talk about drug costs. Yeah, yeah. It's one of the most comical things I've ever seen actually. So let's now talk about this experiment.

I'll tell you from my vantage point, the day I'll never forget, which is I remember getting an embargoed copy on the day before Christmas. So it's Wednesday, December 24th. It's probably noon. It's funny, I was in my office, which is dumb. Why was I in my office at noon on Wednesday,

the day before Christmas? I certainly shouldn't have been, but I remember being in my office, and is dumb. Why was I in my office at noon on Wednesday the day before Christmas? I certainly shouldn't have been, but I remember being in my office, and I remember how sunny it was. I remember what a beautiful day it was, and I remember someone from the New York Times emailing me the embargoed copy, and it's embargoed. Yeah, I was embargoed for another couple of hours. The person at the time she sent it to me knew how interested I was in the subject matter, and I'm reading this thing, and I'm like, this is unbelievable.

I just couldn't believe what I was reading. And I have a background in immunology, so of course I can understand what these figures are showing. Give people a sense of how long it takes to get there. So if the public is first seeing this in December of 2014, when did the experiments start? And I mean that conceptually. Like I don't just mean you're enrolling patients. Like this is about a four or five year journey, right? That's right, about 2010 we started.

So what was the hypothesis that you wanted to test and that you, John, and the team wanted to test? There was some pre-work before we could ask the question. The pre-work was one, we reviewed the existing literature. We were especially that paper from Chen. And we then looked at some other work that had looked at drug levels in cellular systems necessary to partially or fully inhibit the target. And we had to look at a lot of different cells because torque ones biology, while in the big pictures the

same in different cells, the sensitivity of the complex to the drug is different in different cells. And we have some hypotheses now for why that is. And we then did some modeling to understand whether the low doses, and we looked at internal data that the company had to see, could we come up with a low dose and a schedule that would yield exposures in people that would partially or fully inhibit torque one yet give less than assay trough levels

to help ensure safety and the healthy volunteers. Now, Lloyd, was this mostly because at the time you wanted to see if it even made sense to pursue a new molecule entirely that would inhibit complex one or so called selective inhibition, but the idea is why go down that path of doing that without a proof of concept that says it works, or did you think at that point in time, if we can get this to work, you would never need a selective M-Torque one inhibitor. You're asking really for Novartis thinking

that's probably still confidential, but the big picture is we wanna know if something works. We wanna figure out if we can make it better. Again, ultimately, Novartisus we at Restor Bio, and hopefully everybody at a pharmaceutical company is thinking about how can we make patients' lives better? And then everything else is important, but secondary to that.

And then what do we measure? What dose do we use? How frequently do we give it? How long do we treat people? How do we answer the question clearly? What do we measure? All of those questions had to be answered and they had to be answered before Joan brought the program to the decision board of the company to say, give us a lot of money to do this experiment. Each of these clinical experiments cost millions of dollars.

And at the time, Rad 001 was FDA approved for another indication or no? Yes. It was already approved in cancer or in... I think it was in renal silk. RCC? Yeah. Okay. So that only raises the stakes of what you're asking because you're taking a drug that's already gone through phase three

and you're going to spend a lot of money on it that you technically don't need to spend. Is that a common thing to do inside of a company as large as Novartis to take a drug that basically you're trying to make the case for a totally different use? Yes. I'd suspect most companies do things like this where when a drug is registered in one indication, if you can find others, it's a good thing. So, I mean, for the sake of time, I will just, I mean, basically, say that you guys did something kind of amazing, which is

with so little human data, you did a great job of identifying the right patient population, identifying the right patient population, identifying the right primary outcome, identifying a correct power analysis, so you wouldn't miss the signal. So in other words, you didn't know how to power the study, which means you had to have a sense of how much the benefit was going to be,

knowing how long to pulse, how to dose, I mean, this could have gone sideways six other ways. It could have, we had some additional help too at the time of artists at a vaccines group and they had commercialized a flu vaccine. So we knew a lot about flu vaccination and responses required for making a clinical difference and so forth. So let's walk people through the study design. You've got what? About 300 people aged 60 plus more or less.

I think it was 218 in that 214 paper. Everybody was over 65, no unstable medical conditions. And were these subjects on Australia? Was there something about this? It was Australian. Well because this was a flu vaccination, so let's just skip ahead the endpoint of the study, the primary endpoint by which we were going to decide did the study work or not work, was the response to a flu vaccination, the seasonal flu. Now, because it's a seasonal vaccine, we had to do the study when we were ready, wherever

in the world people were going about to get their flu vaccines. In that paper, we were ready, wherever in the world people were about to get their flu vaccines. In that paper, we were ready for the Southern Hemisphere, because our summer is their winter. Right. And since the CVS and the Antarctic ran out of vaccine that year, Australia made the most sense. Australia, New Zealand. Yeah. So there are four arms in this study. There's a placebo arm.

It gets just, obviously, a placebo. There's three treatment arms, one that gets 0.5 milligrams of Rad001 daily. So, ever-olimus. There's a group that's getting 5 milligrams once a week, and there's a group that's getting 20 milligrams once a week. So, it's a clever design because the five in the 20 that are both 20 milligrams once a week. So clever design, because the five in the 20 that are both getting it once a week gives you a great, you get to answer both efficacy and toxicity questions as they pertain to that dose. The point five daily versus the five weekly

is your closest aggregate dose where you get to see is there a difference in trough. So overall, a lot of interesting stuff. What was your personal null hypothesis? Not necessarily the same, but do you recall what your null hypothesis was going into that experiment? I mean obviously the null hypothesis is that there's no drug effect. Yeah, sorry. What was your first alternative hypothesis, I guess? It's a better way to say it. This was a little bit less hypothesis testing the way that academic investigators work than it was asking questions. And the question was in the receivore, but at a high level, it's, can we see an

improved vaccine response at an acceptable level of toxicity that would have this drug make sense. And that's the high level question. And you went through the doses and schedules we used, and we tested three different ones because each of those doses did a different thing to torque one inhibition. The 0.5 milligram dose partially inhibited in a sustained fashion, the 5 milligram once a week fully inhibited torque one for a couple days out of the week. And the 20 milligrams we modeled would fully inhibit torque one over the dosing interval. I didn't realize that actually. 20 is so high that it gave you functionally non-stop inhibition of mTORC 1 until your next dose.

Did any of these have mTORC 2 inhibition? I would have expected the 20 milligram would have. I don't remember that anyone had to discontinue the drug for that reason, but it's been a couple years since I read that paper. Well, let's look at the toxicity table. So table one of this paper, which is again, such an interesting paper, I was surprised. Obviously, it was the first thing I looked at.

Usually table one is inclusion criteria or something like that, but you guys just skip the foreplay and went right to it. Table one, incidents of treatment related adverse effects. I wish I could honestly say I remember how I read this the very first time because I've looked it at a number of times since. But what's interesting is I certainly remember seeing that the placebo group had 21 adverse events.

So that's important to always keep in mind when you look at clinical studies is there's just a baseline level of adverse effects that have no bearing on the drug whatsoever. So it's almost like you could subtract 21 out of all of the others to get a sense of what the noise is. So the group that got 0.5 daily had 35 adverse effects and each of the treatment groups had about the same number. So I mean there were 53 groups in each of the three arms, 59 in the placebo. So the 21, you might discount that slightly.

But it basically went from 35 to 46 to 109. So at this point, obviously, this means each patient is having more than one adverse effect likely. But here's what I found interesting. The next line in the table tells you how many people actually had adverse effects, so not just the total adverse effects. And this was surprisingly constant. So in the placebo group, it's 12. In ARM 1, it's 22. In ARM 2, it's 20. In ARM 3, it's 27. So looks like 0.5 daily versus 5 weekly.

No real difference in adverse effects. And by the way, I'm not going to, don't worry, I won't read you guys the whole table here. We're going to link to this paper in the show notes. The other thing that really stood out to me though in terms of side effects was mouth ulceration. Now, that's the side effect I remembered the most from residency was the patients getting apthosalcers with their daily dose of rapamycin.

And most of them were getting more than 0.5 daily. So, most of the patients that I worked, my recollection was that two to four milligrams was a very common daily dose for rap immune. And again, this is rad 0, 0, 1. So it's a different vehicle, but it's comparable. And so 0.5 daily would definitely be lower than what I was used to seeing people get. And yet 11.5% of these folks had mouth ulcerations, whereas the people getting 0.5 daily, where at 0.5 once a week was about 4%, and 21 so week was about 17%.

So that's really interesting. I mean, that tells a very interesting story about the kinetics of this drug. Was there any other toxicity that surprised you in the study? Well, as you pointed out, mouth ulcers are one of the more common and fairly specific side effects for rapologs. Should we spend one moment explaining why I think we could certainly speculate why? I don't think anybody knows why there's

some hypotheses, but and it isn't just rapologue-associated mouth ulcers. We don't know what causes the ordinary spontaneous apthasalcerations that people get. There have been a fair amount of work on it, but nobody knows. There are a lot of mysteries in medicine, and that's just another one of them. Anacthodally through five years of residency, I don't think I went more than two weeks without an apthasulsar. They can be stress-related. I'm sure they are, and it was to the point where they would drive me so bananas. I couldn't even get relief from those sort of topical lidocaine gels.

The only thing that could give me real relief was if I could inject bupivocaine directly into it because it was such a long-acting agent lidocaine only lasts an hour or two. That was not gonna do me justice on a call night. And it got to the point where I would sit there and inject bupivocaine into my tongue or into my mouth. And I remember once somebody walking in the call room while I'm sitting there holding my gums out, jamming bupivocaine in, and I'm kidding them.

They must have thought I had some drug problem. But two weeks after leaving residency, I never had an apthas ulcer again. I hope you never have another one there. I'm very pained. They're unbelievable, but then of course I did get them once I started rapamycin. So we'll come back to that. But I certainly went many, many years without them again. My hypothesis was some combination of stress and sleep deprivation might have played a role.

It doesn't help mechanistically though. We just don't understand them. So overall, I thought the side effects were less than I would have expected. Let's now talk about the results. I'll actually hand you the table here because I don't, not that I would ever expect you to remember table 2A, but walk a little bit through kind of what you guys saw

and how you tested it. So you're using a couple of different flu vaccines, et cetera, a couple of different strains. Yeah. So the standard flu vaccine had in those days, three different antigens for three different kinds of viruses, two influenza A strains, one influenza B strain. And I think as we all know, the flu vaccine is designed every single year based on the circulating flu strains in Asia to try to match the vaccine to the strains of flu

that we expect to get. In this case, in the Southern Hemisphere or for us in the Northern Hemisphere. So patients were treated with one of those doses of Rad001 or placebo and the study was randomized, double blind and placebo controls. So neither the doctors know the patient's new who was getting what. And I guess there's one detail I omitted which if my memory is correct, the patients were treated for eight weeks, six weeks, six weeks, and then there was a washout.

So then they had a period of nothing for, was that six to eight weeks? It was two weeks after the six weeks of treatment. Oh, that's where the eight came in. So it was six of treatment, two of washout, then vaccination. Right. And the rationale for that was we wanted the drug to be completely gone. At the time we vaccinated. So we were asking the question, is this a residual effect of the

drug on the immune system not a lingering effect on the immune system, per se? Yeah, not a direct effect of the drug, but an indirect. Yes. Okay, so with that said, what was the first finding? So, we were looking for whether the response to the flu vaccine was 1.2-fold better in a drug-treated group than the placebo, and that 1.2-fold came from a previous study that had been published, that showed that was the minimum requirement to see a clinically meaningful decrease in symptomatic flu in vaccinated patients. We saw, and we required, and this was pre-specified, two out of the three strains to have that improvement. And we saw that improvement, or better, in the two low doses of Rad001,

but not in the high dose. And the high dose we just saw one string better. One of the three strings was better. And the other two were actually a little below one. So a little below the placebo. What do you make of that? Is it noise? Do you think there's something mechanistically happening there? I do.

I think we certainly know that high doses of a rapologue are immunosuppressive. The dose of 20 milligrams once a week is sufficiently high to fully suppress torque one. I expect that we probably interfered with lymphocyte proliferation. And do you think it's just a tweak that you didn't see? What's confusing to me is that you still saw a much greater immunity in one strain. And if I recall, it was even higher than the two low doses, thinking about the first figure in the top figure in the second figure of the paper. So that would be, my first guess would be, oh, clearly you just hit the daily dose of

an immunosuppressed patient if they were all below baseline. But if you did a 25 weekly in there, do you think you would have just seen them all eventually start to go down? Certainly if we got to some high enough dose, I think they all would have been low. I got it. Yeah. So it's almost like there's a J curve here. They're an upside down J curve really. An upside down U, I suppose, of some combination of dose and frequency producing a sweet spot where we're seeing. And by the way, I can't recall it's been so long since I looked at the paper. Was there any lympho proliferation?

I mean, these were functional assays. Any changes in the counts of lymphocytes or any other... This specific assay that was the primary endpoint was in anybody-tight or assay. Right. We didn't do mixed lymphocyte reactions or some proliferation assays part of this paper. Did anyone ever look at fractions of lymphocytes, for example, did anyone look at CD25, CD3 to see if anything had happened to suppressor T cells? We didn't do it in this study. Well, we didn't do functional lymphocytes

acids. Part of this study was a very comprehensive multi-dimensional phycidometric assay to get lymphocytes subsets. And we reported one of the results in figure three, which is where we saw improvement in checkpoint protein levels on both CD8 and CD4 lymphocytes, which mean and checkpoint proteins are very popular in oncology now. And some of your listeners may have heard about those things like PD1, for example, the drug target of ketruda or optiva. We had ketepherity on recently and we had a beautiful discussion about checkpoint inhibition, but that said, let's assume people don't know what that is. It's always worth redescussing it. Yeah, these proteins inhibit lymphocyte function and they go up as the lymphocytes get exhausted. And what we saw in this study is that the level of PD1 went down on the lymph sites

in the drug treatment group compared to the placebo. By what percent? It was a relatively small effect, a 10 to 20% change. But comparable to the effect that you saw in the increase in antibody recognition. I mean, it was like the mirror of that. I guess, yeah.

And presumably the teleologic rationale for that is the more tired olympicite gets, the more it wants to weaken its brakes. I'll go along with that. Everything in me in my world Lloyd comes down to just anthropomorphizing the immune system. That's how I do things. So, has anyone looked at this by the way to see what rapper Myson does or rapologue does in this type of intermittent dosing to inhibitory T cells? There have been a lot of studies of T cell subset functions with rapologs, both in mice and in humans, looking at effector memory transition, looking at T-regs with high exposures

or substantial inhibitory effect on B cell function. There's a lot out there. Do you think that rapologues could be used to suppress T-regs? Selectively, of course. There are a couple of papers that show that. Because it would sure be interesting to start layering in rapologues with immunotherapy. Uncology specific. Yes. Yeah. There's a small literature on that. What else did you see in this paper? I think we've touched on the high points. Yeah. The other experiment we did, which I think we mentioned in the text, but didn't go into great details, is the flu vaccine is a T-dependent antibody response. We also vaccinated patients with a 23-valent pneumococcal vaccine, which is a pure polysaccharide vaccine, so it's a T-independent response.

We were thinking, could we improve antigen presentation, perhaps if the dose of the rapologue we used augmented autophagy, and could that contribute to antigen presentation? We measured seven of the 23 antigens. Wait, tell me why that would be, that's not an obvious purview into a topogy to me. So let's back that up a little bit. So you're giving them a pneumococcal vaccine, which is what type of vaccine? That's a polysaccharide antigen. Polysaccharide antigen, you're not giving back the whole bacteria, and how does it get presented to the immune system?

There are specific elements of innate immunity that recognize bacterial polysaccharide antigens, and that brings it to professional antigen presenting cells, likely dendritic cells, then it has to be internalized and then presented. So that's MHC class one. This is an peptide presentation. This is presented in the context of in a net element that recognizes polysaccharide antigens. Okay.

All right. So we're outside of class one class two. Yes. And you're saying the ability to internalize or basically the ability to phagocytose that like papalis accharide and then and directly stimuli B cells. Yeah, it was an exploratory element of the study we saw in the seven specific antigen responses we measured if I recall six of them increased, but by a small amount. It was an encouraging trend, but we didn't further follow it up.

Okay. Any other markers you could have for your forotophagy? Did you look at light chain transition or anything like that? No. Autophagy is a really difficult clinical investigation paradigm. And I know you recently saw Mitch Weiss' paper on unpaired hemoglobin in thalassemia patients. I was super excited when I read that paper because here is now a clinical paradigm where we can test drugs that augment autophagy and see something easily measurable in a not that rare patient population. Yeah. Let me think. In those patients, did you collect serum that allow you to look at amino acid levels in the plasma or anything else? No, we didn't do any of that. Do you still have any of that bank to serum?

I don't think so. Easy enough experiment to repeat, but of course, the other thing I'm curious about is, is there anything about Rad001 administration that mimics fasting, for example? How much of the benefit here is through direct inhibition of MTOR, and are there any pleotrophic benefits that aren't quantified through this? Obviously, some of them, the fact that you waited until the drug was out of the system to check the immune response is actually a great insight, because obviously you eliminate some of those things.

But in the way that many people have argued statins have a direct effect, they inhibit cholesterol synthesis, an indirect effect, the liver up regulates LDLR, a really indirect effect, which is immune suppression or other sort of benefits around endothelial health and things like that. Do you think there's a possible third leg to this stool that we haven't thought about? The fasting story is kind of complex. So do I think there's some other persistent benefit of a rapologue? I think the mouse experiments tells us there is, and it's because relatively

short course of a rapologue is nonetheless sufficient to extend a mouse's lifespan. And we do not understand that. Although it's so hard to extrapolate what short-versus long means in a mouse, isn't it? Yes. Look at the mouse that fasts for 24 hours. Look at J. Mitchell's stuff where they do a one-day fast prior to a femoral artery ligation and a reperfusion where the mouse that was just fed normally through the insult, they all die, the mice that had 24 hours of fasting prior to a lethal reperfusion injury, either all or mostly live. I don't know how to extrapolate that

into a higher order animal, because it's not even the duration of the fast, it's the metabolic consequence of the fast. There's some long-term consequence of that that we don't understand. And there's several things you could hypothesize. Is there a change in the DNA structure based on histone methylation or DNA methylation? Is it, or is it something else? Those are just the

things that come to mind. Yeah, that's a great point, right? Is you could literally be resetting methylation on that. You could turn back a methylation clock to its template, potentially. But people have looked at that with rapologues and it doesn't seem to happen. In other words, you take a whorevath clock pre-imposed and you're not seeing unwinding of methylation. Yeah. That's been done in mice as well or just in humans. I suspect it's been done in a bunch of species. It's one of these sort of negative studies that may never get published.

That's a pet peeve of mine, by the way. Negative studies not getting published. I think it's a pet peeve of a lot of people. It's hard enough to publish positive studies. And I'm sympathetic and agree. So we're high-fiving on New Year's Eve 2014. How do you go from... What is no artist? Again, if this is confidential, I mean, we'll

skip it, but what are the brass at Novartis think of the results of this experiment, which is effectively taking a drug that we already have on the market for a very clear indication and now potentially expanding an indication, can the FDA take a study, this is a very well done study here, this is double blinded, this is placebo controlled, and this found a significant outcome, is that enough to change the indication for a medication like this? I can speak in general in that for an indication that could be relevant to many, many people, you need a corresponding, corresponding amount of safety data. This study was way too small for studies that would be required for marketing authorization.

I got it. So because something like renal cell carcinoma is relatively infrequent relative to influenza vaccination, you had enough safety data to justify treating people with RCC. This would not constitute sufficient safety data to basically give every person over the age of 65 who's getting vaccinated this type of medication. That's exactly right. If we fast forward a little bit in the conversation, we've advanced this program in Restore Bio, Novartis licensed it to restore bio. And our phase three program is two very similarly designed clinical studies. One has a thousand patients.

The other one will have about 1600 patients. You able to speak about how the decision was made for Restore Bio to basically acquire a piece of an asset from Novartis and what else was brought into great that company. And how did you, Joan, and I assume many others decide to leave? I mean, that's obviously a loss to Novartis, presumably, which implies that they probably still have invested interest in this excessive restore bio.

Yeah, let's take one step backwards and answer an earlier question you asked, which is, what did Novartis think when we got these results? Oh, yes, yes, thank you. And I think everybody was very excited for reasons that you're excited. That was my Christmas present of the year. And the guidance was, this is so important. Let's go back and do it again, try to do it in a more torque-1 selective way.

Are you able to say how much that study cost just to give a sense of? I don't think we talk externally, but any clinical study like this phase two study with two to three hundred patients cost millions. A lot of the cost is driven by often exploratory assessments you do in the context of the study, beyond the per-patient cost and investigator cost. But millions. I mean, it's a lot of money for anybody, and it's done with a lot of deliberation and thought.

So the guidance was to go back and do it again, make sure it's real, come up with a way to be more torque-wise selective. But using the same vehicle, which means putting a finer point on the dosing, not necessarily, I mean, you're still not at the point where people are saying, we need to make a new molecule to replicate this. Well, we had one. Oh.

The research team at Novartis had come up with a very cool finding that a combination of an allosteric and catalytic torque inhibitor could be more torque-one selective and more potent. There was actually synergy. This is published by Bayott Nyfeller and Lone Murphy. They showed synergy, so a rapologue. Can you explain to folks what the difference is between Alistairic versus catalytic inhibition?

Sure. So catalytic inhibition means an inhibitor that's binding at the catalytic site of torque one and blocking phosphorylation of targets. An Alistairic inhibitor is a fancy way of saying an inhibitor that binds someplace else on the molecule and nonetheless inhibits it. Rappamysin, come back. I always think of Allisteria as sort of shape blocking. Could be.

Let's say shape blocking. So in this case, the Allisteria inhibitor is the combination of FKBP12 plus Rappamysin binding to torque one. So the combination of those two synergistically inhibits torque one and It's a little more torque one selective Everolumus does the same thing. It also binds to FKBP. Yes, it binds to FKBP's also Maybe later on we can get to that laming paper, which is which yeah, I'd love to get to that paper. Yeah, yeah so with that understanding, we could then explore, put a finer point on the dosing as well

as explore the biology of that catalytic inhibitor with and without red 001. And that's what the next study did. Overall a very similar study design, we treated for six weeks, two week washout, interrogated the immune system function with flu vaccination. And got fundamentally similar results. There's one other point I'd like to bring in because it leads to where we are now. In the very first study we did, in analyzing, we saw immune function improvement, which, and our marker for that, was flu vaccine response. In the adverse event listings, we saw fewer infections in the drug-treated

people compared to the placebo patients. Over what time, Horizon, we followed people for a year. And by the way, did that increase in vaccination translate to a reduction in influenza, or was that the infection you're speaking about? Are you speaking about all infections? All infections. Okay, what about influenza specifically? Two few events to be able to make a conclusion. It's underpowered to look at the flu.

Exactly. If you think about all infections you get, or patients get, most of them are respiratory tract infections, colds and flus in the winter season. An interesting thought experiment, you wouldn't do this experiment, but an interesting thought experiment would be a two by two, vaccine, no vaccine,

RAPA, no RAPA, powered to see difference in infection. Big experiment. Huge experiment, but great, Gadonkin, right? Yes, I agree. So in this very first study, we found, again, we weren't thinking about it and we weren't looking for it, but we observed it in the adverse event listing.

So what were some of those infections, Lloyd, that you sell, like, UTIs or cellulitis, that kind of stuff? Two most common ones. By far, the most common was upper respiratory tract infection or respiratory tract infection. That was not influenza, yeah. Maybe some of them were. We didn't measure.

We don't know what the pathogens were. We do know from surveillance experiments that the CDC has done that most of them are rhinovirus and then there's metanumovirus. There's various other echo, God only knows what. Yes. And we saw a decrease in UTIs also. And that persisted for a year. The biggest effect was when the patients were shortly

over the course of drug treatment and some time thereafter. But even if we analyze it over the course of a year, we saw it, although the effect waned. In this very first study in 2014, we brought patients back a year later and re-vaccinated them to see if the improvement in immunologic function persisted and it did not. Okay, so by a year they'd clearly lost it. Yes.

And of course, we didn't do the experiment, but do you know if six weeks was necessary, or could you have done four plus two or three plus two or two plus two? Would the benefit have been better if you went eight plus two or ten plus two or twelve plus two? Like how did you agree on six weeks of treatment? I understand the two week washout, but what about the six weeks of treatment? It came from two places. One is that that's what they did in the mice. My same caveat implies. Totally agree. And then secondly, we know the timing of lymphocyte production from committed precursors in human bone marrow.

And we were thinking that the drug could be acting at that level. And six weeks of treatment is sufficient to, by the time you've accinated eight weeks have some new lymphocytes from those committed precursors So your hypothesis would be six was the minimum time required to get a full turnover and going eight versus ten Would not necessarily bring benefit and might only expose you to longer side effects We actually never tested this. We came up with our dosing period for the rationale that I gave you, and we've not, at least with a rapologue, tested other intervals. We did see in the second study, which also worked on the vaccine endpoint.

We promoted respiratory tract infections and infections in general to secondary end points. So we're looking at them prospectively. We're collecting them more carefully. That was in the second study. Yeah, in the second study. And again, the drugs decreased the incidence of respiratory tract infections. The biggest effect was observed with just the catalytic inhibitor alone, the second biggest effect. Wait, the catalytic inhibitor alone, the second biggest effect.

Wait, the catalytic inhibitor was a new molecule? Yes, it's a new compound. In that paper, it was called BEC235, and this is the molecule licensed by Restorbio. So BEC235 is not rad 001 combined with something else. No, we tested the combination, and it also decreased respiratory tract infections and the combination was the best at improving the flu response. But the single catalytic inhibitor was the best at preventing respiratory tract infections.

The study had an extra arm. It was a somewhat bigger study, 264 patients. Was this the one published two years ago? Yes, 2018. Oh yeah, yeah, yeah, you're okay. So let's now talk about the creation of restore bio. So essentially, the story is that we did the second study for prevention of respiratory tract infections and enhancing immune function with hemorrhidabation, worked again.

enhancing immune function with MTOR inhibition, worked again, everybody's excited, and Novartis, as in most big companies, big pharma companies, the research teams produce more than global development can handle, and it's done deliberately. It gives early and drug development, you never know exactly what's going to work best. Want to create enough opportunities so something will be exciting.

And the program transitioned to global development and they had a lot of exciting things to do in this feliball of the funding line. It reflects in part the excellent productivity of the Vartus research. It reflects in part the great opportunities the global development has. Of course, as the champions of the program, we were kind of disappointed. But Novartis felt, and again, I'm speaking for myself now, I'm not a Novartis spokesperson,

but Novartis felt this drug looks like it could work, and it could help people. We have to find a way to make it available to people if it could work. So other pharma companies do this too. The decision was to out license it. And Joan, as the sort of originator of the idea and the biggest champion, wanted to go outside and have artists and start a company.

I introduced her to an absolutely awesome CEO I know who was ready for his next role and They raised money and they created a restore bio and that was a pretty quick path to going public Restore bio went public late in 18 didn't they it reflected in part the Need for funds to run a phase 3 program. Yeah, what did restore bio raise in the IPO? You're asking me a hard question. I was at Novartis But probably around 90 million I think wow. Yeah, so Yeah, as you said that's you're basically going right to phase three at this point because so you licensed one or two molecules from Novartis restore bio license be easy to three five, which is now named RTB 101 and They then did a phase 2b study.

These two studies we had done were phase 2a. So in the first study published in 2014 that we've been discussing, respiratory tract infections and infections in general were observed to be decreased in the drug-treated group compared to the placebo by reviewing the adverse event listings. This was not something we had considered our pre-ore. But we recognized decreased infections could be a consequence of improved immune function, which

is why we looked in the first place in the listings. And then in the second study, where we explored a catalytic inhibitor, which is now our TB101, RAD001, as well as the combination, we promoted respiratory tract infections and infections in general to a secondary endpoint, and we specifically included collecting those data both by patient reporting as well as investigator querying the patients at home regularly. And again, we saw a decreased respiratory tract infections. The best drug was BEZ235, which we now call RTB101, in terms of decreasing respiratory tract infections.

The combination also worked. Interestingly, the combination, as well as RAD001, improved the flu vaccine response, but the BEZ-235 or RTB 101 did not. Remind me, it's catalytically inhibiting. So it's binding directly to Tor. Yes. And is it binding equally to Tor when bound to Raptor, meaning complex one as it is binding to Tor bound to Richter known as complex two. is in a cellular assay context, so we don't look specifically for the binding, but we look for inhibition of phosphorylation of sites of serine-6, or which one, S6 kinase, for torque one, and phosphoakate there.

And we look specifically at the phosphorylation site that torque two does, not the one that any other enzyme does. So we're able to see that a catalytic inhibitor at high concentrations will inhibit both. RTB 101 has some preference for torque one, and it's a little different depending upon which cell you look in. I wanted to come back to that because, and I got to remember where we are in this story because I don't want to lose this thread. So maybe we can agree to just park this again, but I definitely don't want to leave the discussion of tissue selectivity.

We've focused so much on C1, C2 selectivity, but we haven't talked about muscle versus liver versus adipose tissue, for example, which you could argue, you might want very different behaviors there. In terms of drug design, drug pharmacokinetics, is it easier to target tissues or is it easier to target enzymes, proteins, et cetera? When designing a drug or waving magic ones, no magic ones involved.

It's far easier to design a drug to hit a target. To hit the target in a particular tissue is harder but doable. Sometimes you can do it in a deliberate, designed fashion. For example, making a pro drug that's cleaved to an active form only by an enzyme present in your desired tissue. You also have to be mindful that within every

tissue there are multiple cell types and you want the drug in the cell type that will make the difference. And again, all of this is possible and it's just how much time and effort you're going to put into it. So when you go back to even the first experiment in 2014 and all of the animal data that came from it, did you have a sense of where this was acting tissue wise? Did you feel like you were acting on bone marrow? Did you feel like you were possibly acting in the thymus? Did you feel like you were acting in some other cell line that directly or indirectly was playing a role.

I mean, or did you have a sense that you were seeing this everywhere, but it didn't seem to matter except in the bone marrow. We thought it would be bone marrow and perhaps secondary lymphoid tissue, which are lymph nodes or glands, but we didn't explore it exactly. In general, small molecules that aren't specifically tissue targeted will often go to many tissues. We knew from toxicology studies with the compound that it distributed to our target tissues. We felt we had enough information to move ahead. Yeah, so when RTB 101 was basically the basis upon which restore bio is formed, correct?

That's right. Now, I was very confused during your road show, which was about a year ago, maybe more than a year ago. It might have been early 2018, I've sort of lost track. You were at Novartis at the time, so it wasn't really your road show at the time, but I naively, I guess, thought that RTB 101 was actually rad 001, the Evalymas, combined with a PI3K inhibitor. So, that's actually what I thought was happening.

And I remember even having discussions with other people looking at the data and saying, is this what this company is? So, I assume that this company outlicensed Evalymas, combined with a PI3K inhibitor. So, how were we confused by that? When RestorBio was formed, it licensed RTB 101 from the Vartis for all uses. And there was also a limited license to use RAD001, only in combination with RTB 101 for our indication. Now the phase 2b study that, restored by O'Ran, showed that the most effective drug or drug combination for preventing respiratory tract infections was just RTB1 will one alone

at 10 milligrams. And remember the previous study that Novartis had run had shown that although the combination was best at augmenting a vaccine response, it was just RTB1 or 1 alone, which at the time was called BEZ235, was the best at preventing respiratory tract infections. We believe this is because the mechanism by which it prevents respiratory tract infections is upregulation of an interferon stimulated antiviral gene response. Interferon, remember, is a substance in the blood that upregulates many different proteins, most of which are involved in preventing viral infections. And you need protein synthesis to make

all of these proteins. And I worry that if we inhibit M-tore for a long time, we can upregulate the genes, but they won't be expressed adequately. And there are some literature data that you need emitter in order to express the proteins induced by interferon. So these experiments taken together suggest to you that, I guess it just reinforces this idea of intermittent dosing, not just intermittent within the week, which I think was clearly established by the Phase 2A study, but even applying a secondary cycle

over the course of a year, for example. I mean, you know that doing a six plus two once a year is probably not adequate. So there's some frequency upon which you wanna met a cycle that. But the reason you don't just wanna go all the time taking it presumably is you might actually start to impair protein synthesis that's necessary for lack of a better word, basically empower your new superpowers

of immunity through enhanced protein synthesis. Yes. Protein synthesis, there's several different kinds of protein synthesis and some are more or less sensitive to inhibition by rapologue. Which, white is very recent paper. One of the interesting things I found in it was that there wasn't much of an inhibition of hemoglobin synthesis, despite the fact that they were using fairly high exposures of a rapologue. So I think there are some proteins that are sensitive

to translational inhibition by rapologues and perhaps some that are less sensitive. Before we go down this path of getting a little bit more into RTB 101, let's take a step back here and say, do you think that all of the benefits that we saw in the ITPs across all these other species? If you think of the benefits that Matt Cabralin is seeing in dogs, if you think of sort of the global excitement

that exists around rapamycin and rapologs, how much of it do you think is mediated through what you guys are testing, which is you're clearly enhancing immunity in a positive way, which could have at least two very distinct benefits. One is the reduction of infection. The other could be frankly reduction of cancer through increased surveillance. They're very similar viruses and cancer obviously behave or susceptible to the similar branch of the immune system. Do you think there are other things that we haven't talked

about yet, such as increased the topology, targeting of end-door destruction, end-door, desilencing of synescent cells, reduction of inflammation, enhanced mitophagy, what other mechanisms do you think could have been involved here and what evidence exist to support or refute that? Well, I think we know from academic experiments that every single one of those mechanisms can extend health span in preclinical models We do not know in people and I think Similarly to follow up our earlier discussion about what tissues you have to inhibit MTOR in in order to get a clinical benefit

We don't really know the answer to that either. It's been studied in some of the preclinical models I can recall an experiment in the Drosophila fat body where inhibition of MTOR right there was sufficient to extend a fly lifespan. There's still a lot we need to learn. What does your intuition tell you? How much of an overlap or parallel do you see between the benefits of fasting and caloric restriction

and the benefits of rapamycin globally. Yeah, so one of the interesting things that we did and was done previously in a nature publication, I think the author was Sanghupta was looking at the consequences of fasting on emtore activity. In young people, as you would expect, fasting leads to suppression of emtore activity, activation of the cellular recycling machinery, autophagy, suppression of protein synthesis and DNA and lipid synthesis and so forth, basically preparing for lean times. An old mice that's impaired. We only done the liver tests in mice. So we back translated this experiment and gave,

actually, I think it was rats. It was all rats, doses of mTOR inhibition that corresponded to the doses we were using in people that were well tolerated. And then we looked at their ability to suppress MTOR. So, in the old rats, even with fasting, their MTOR was still active in the liver. In a young rat it's suppressed. So, the young versus old had the same degree of inhibition to the same dose of rapamycin. Well, you couldn't test in the young rats because their emtora was already low. Oh, but if you did it outside of the fast, I mean.

Well, certainly the exposures were the same. There was no age-dependent difference in exposure of the liver or the blood. That's interesting. Does that suggest that the older animal lost the ability to respond to the environmental reduction in nutrient? Exactly. Exactly. Hmm, that's upsetting. Although it does explain a very interesting finding, which is everything comes back to the 2009 paper. What really was interesting scientifically was that those mice were 600 days old. Those were

mice that if you fasted them, wouldn't have lived longer. It already passed that stage where caloric restriction would extend their life. And yet their lives were extended 15 and 25% by Rapa Mison. That was a big, freaking deal. Yeah. We published our experiments in that 2008 paper. It was sort of an interesting back translation experiment where we treated the old rats based on what we do about the old people.

And of course, we could do in rats. What we can't do in people is take their livers out and study their emitter inhibition. But we weren't the first ones to do that. There was a very good nature paper that showed the same thing. Do you think this applies to humans? I mean, do you think that intermittent periods of caloric restriction are not beneficial to people in their 60s or 70s, which would be the equivalent of those quote unquote, old rats? The only thing that our group has been able to try is we looked at whether

we could detect mTOR activity as assessed by things like phospho-six kinase in the peripheral blood leukocytes of old people, and we couldn't detect activity. We can't answer the question. I think we would need liver samples under fasting conditions. I mean, are you volunteering? Yeah, I'm absolutely volunteering.

No, I tell you, there's a lot of things I subject myself to. I'm never excited about the liver biopsy. I just... I think that's the problem of doing a residency in general surgery is you've had one too many calls down to the interventional radiology suite with the patient that you have the recency bias of you only remember all the cases of liver biopsies gone bad. All those hepatologists that have never had an issue, you don't hear about those cases, but you hear about everyone that there's sort of a referral bias. You never see the thousand that go well. You only see the one that didn't. I don't know. I think at some point, I'll probably have to sign up for a liver

biopsy. I think there's a lot going on there. There's so many questions I have about the liver, especially my own. No liver biopsies. You can get samples other ways, at least for this reason. But it is an interesting open question and yet another one of these things We don't know is is mTOR suppressed in elderly people with fasting and in which tissues and by the way Do we know if a topogy is impaired in older folks with fasting? Because atophagy and mTOR inhibition are not synonymous. That's right.

They overlap, but they're not synonymous. Yeah, well, there's a lot of biology there, and it's not only mTOR that can trigger atophagy, there's other mechanisms. There's Beclin-1 mechanisms and so forth. But it's an interesting set of experiments to do with a young group of patients and an old group of patients.

And there's a priming effect to this that I just don't, I mean, it's so multifaceted to study all of these things. You think of the infinite combinations you can have, which is what's the effect of Rapa plus fasting when staggered, for example. Does one prime the other? It's hard, you can't really go on phishing expeditions with these questions.

You have to be more thoughtful in your hypothesis generation. There's just too many variables. That's right. There's too many ways to be fooled. That's right. So what can you tell us now about RTB 101? What has been published on this? In other words, I don't think we can speak about obviously anything that's not published

at this point, or at least hasn't been publicly signaled. What's next for this compound? So the excitement is in the Phase II A study that Novartis ran, we saw decreased respiratory tract infections in elderly people treated with it. In the Phase II B study, that restored BioRAN, again, the same dose 10 milligrams once a day saw the same thing, a decrease in respiratory tract infections.

Now, that study was a complicated study, and did it also have an RTB 101 plus rad 0, 0, 1 arm? It did, and there was not a decrease in respiratory tract infections there, but there was an increase in immunity, or was that, that was a secondary outcome. That was assessed, but it hasn't been talked about yet. The cool thing about the 2018 paper that was published from the Nevada study is that because we saw a decrease in respiratory tract infections, but we did not see an increase in vaccine response, it told us that the mechanism for the decrease in respiratory tract infections had to be something

different. And we had collected some samples for exploratory profiling. We learned that there was an upregulation of antiviral gene expression in peripheral blood leukocytes. So a set of interferon-stimulated genes responsible for antiviral activities was upregulated.

So we identified a candidate mechanism gene responsible for antiviral activities was upregulated. So we identified a candidate mechanism, and it makes a lot of sense. So to put that in English, it's not that the response was mediated by better recognition of viruses. It was mediated by more efficient targeting of and-or disposing of viruses. Perhaps another way to say it is that the vaccine response we were measuring is the primary endpoint was a lymphocyte acquired immunity measurement.

So in other words, you're immune the flu because you've been vaccinated. If you've been vaccinated for rabies, you're immune to that. I've never been vaccinated for rabies. I'm not immune to rabies. In contrast, there's something called innate immunity, which is the immunity of our species. This is an immunity that was developed because we have all co-evolved with our pathogens. And those of us who are here, this is why the LPS on strep is you don't need to be vaccinated to recognize it.

Exactly. So, there are many, many, many other things that we can recognize, elements of pathogens, so we can mount an effective immune response, and we're born with this. We don't have to be immunized for it. And this part of the immune system is what MTOR inhibition can also activate. Yeah. Which is to go back to historical, that's not what we care about in transplant. No.

Because in transplant, you certainly didn't, we didn't evolve to reject kidneys. We evolved to accept our kidneys. Therefore, we reject someone else's kidney. That's MHC-based. Right. Although blood type matching of your organ transplants is for... Well, that's true And now they're doing so many ABO incompatibles that it's I mean the immunology involved in organ transplantation today is remarkable

A subject for another time. I know and I'll start it that you're on the monitor. That was the Nobel Prize right there, right? That's right, Dr. Murray So Is there anything else you can tell us because this is obviously something like have you guys spoken about what the phase three is going to look like? Yeah, well, we're almost halfway through, halfway through enrollment. We enrolled the first phase three study fully and getting ready to start the second. So tell us about the first one. Yeah, so well, let's do a little more on the phase two B because that study answered several questions in one study. We enrolled patients with pre-specified

comorbidities and pre-specified an analysis of them independently. We did doses, we did five milligrams and ten milligrams, we did a different schedule, we did ten twice a day, and we did a combination with a rapologue, our red 0-0-1, with the primary endpoint of decreased respiratory tract infections. We also extended the dosing period to cover a winter cold and flu season, so now we're dosing 16 weeks. Uninterrupted? Uninterrupted.

Okay. Although, with the once-a-day dosing, which is where we saw efficacy, we're only inhibiting m-tore partially for part of the day. By the way, if you had to speculate going back to the very, very first, the 2A with Rad001, if you had taken all four of those groups and measured them at the end of six weeks and then after the two week wash out, what's your prediction as to how they would have differed? I'm sorry, because we didn't vaccinate until 8, if we've vaccinated it at six weeks versus vaccinated at 8 weeks.

Correct, and you did comparison. In other words, I'm asking on drug versus off drug. How much? I know why you had the wash out, but is it also possible that on drug you would have the same immune response? Yes, on the low doses, on the high doses, I bet we wouldn't have. And do you define high as five and 20? High as 20. Okay. Got it. So you think the 5 and the

0.5 still would have had benefit on drug. 20 probably got it benefit. In fact, that might explain the question I asked, which is why did they still at least have one good strain response? It could have been that the two weeks off gave them recovery. Could be. Yeah. So back to restore bio, we figured it. And then the other element to the study is we ran it in two different cold and flu seasons, one in the southern hemisphere, one in the northern hemisphere, 652 patients in the study, because we answered a lot of questions. We found that some patient populations responded well over Over 85 patients and patients with asthma.

Patients with diabetes also responded. Patients who were smokers or had COPD did not. There are some preclinical data that provides a mechanism for why this is the case in the sense that it's a different mechanism for airway inflammation and smoking and COPD and it's exacerbated by amtore inhibition. Oh, so I thought it was going to be a different way of antigen presentation or something. I don't think so.

Okay. The coolest thing about the study is that we saw the same degree of efficacy if we looked at the patients in the southern hemisphere as in the northern hemisphere. It's almost as if there was two substudies in the study. Now each of the patient groups by themselves were insufficiently powered to get any statistical significance, although overall, the patient population did. That's a bold study design move.

That could have backfired badly, right? Because if you had discordance between the northern and southern hemisphere, you would have been underpowered. Well, the goal of the study was to look at the overall patient population, which we did, which included responders and non-responders. And we saw a 30% decrease in respiratory tract infections. Yes, but you had two different strains of the flu, didn't you? Flu was not involved here, it was no vaccination in this study.

Oh, sorry. I mean, what I mean is you had two different environments of viruses. Yes, absolutely. Absolutely. So you deluded, I'm just saying you loaded the deck against yourself. If you did everything identical, but they were all in the same country, presumably you'd have a higher concentration of pathogen. You'd have a better chance of seeing a signal, is sort of all I was saying. Yeah, I think you'd have a higher concentration of pathogen. You'd have a better chance of seeing a signal is sort of all I was saying. Yeah, I think you'd have a higher chance

of seeing consistency. That's right. And you had the lowest chance imaginable because you spread out across two hemispheres. Yeah, we saw consistency. Yeah, so I think we're saying the same thing, which is it's more a credit to the finding. Yeah.

And I'm just saying, thank God. Well, if the drug works, this is what we should have seen. Yeah, it's just a big bet for us to start up to take. And so now we've seen 10 milligrams of RTB 101, decreased respiratory tract infections in the phase 2A, and in each of the parts of the phase 2B, and we use the phase 2B to power the phase 3 program. So the phase 3 program is and the primary indication is respiratory infection or all infection,

respiratory tract infections. And patient population is 65 and up, 65 and up. So the first study, we're calling the program the protector program, the first one is fully enrolled, the 1024 patients. They're getting 16 weeks of drug treatment and we're following them for respiratory tract infections with an electronic record that the patients fill out. If we were just targeting this at Northern Hemisphere patients, is it your view that the optimal 16 week window would be sort of May, June, July, August.

How are you deciding when? Winter cold and flu season. Is when they actually want to receive the drug? Yes. Okay, so basically, so you're so confident that 10 milligrams is not too high that you're willing to give them the drug during flu season or during winter cold season. Yes. All right, so let's turn over to something else you brought up earlier, which was the, the

landing paper that came out about two months ago. That's a prolific lab. Laming was a post-doc in David's lab, so no stranger to this science. There are lots of folks out there that are working on selective mTORC-1 inhibition, not with thinning the potential ways around it through intermittent dosing or looking at catalytic binding or things that might be off you a little bit more insight. What is your take on the biochemistry of selective binding and selective inhibition more specifically? The binding is quite selective. Yeah, I thought basically to summarize that paper

for the listeners, the LAMMING group looked at, I think it was 90 different rapologs, presumably related to rapamycin, and looked for their ability to be selective for TORRIC-1, even with more sustained exposure, then they identified a couple that were, and most of the paper was on one that they liked the best. The really cool thing, and this is going to get us into an immunofilm discussion, is that they found possibly the reason the compound was selective, was that it bound to one of the immunofillons,

but not another. So specifically, it bound to FKBP12, or at least FKBP12 was required for the compound to have activity, but another FKBP, FKBP51, was not. But I still don't understand this. If you bind to FKBP12, and then the RAPA log plus FKBP12 binds to MTOR. Don't you still get into the same problem where after a long enough period of time, you don't have enough TOR to make complex two? I don't think that's why complex two is inhibited. What do you think RAPA MICE and specifically is doing to inhibit complex two then?

I think it's a downstream and indirect sort of counter regulatory signaling mechanism. I see. So it has to do more with sort of the Syrian kinase or the 4-EBP1 or something like that. Something downstream of a direct phosphorylationulating or yes, yeah, I see. And you're saying presumably if you only bind a rapologue to FKBP12, you somehow don't hit that target? Well, I think what their data say is because the compound that they show has no torque to activity at all does not bind to FKBP 51, or at least that's the implication because down regulating FKBP 51, which I think they did with an SIRNA, had no effect on its inhibitory activity, suggests that there's a complexity to the complexes formed, sorry

for that, that we don't yet understand, and it's an exciting area to explore. So, remember, every cell has many immunofillons in it. It has several cyclophillons. It's got several FKBPs, so FKBP 12, 51 and 52 are probably the big three, but there are a few others. By the way, I thought RAPA only bound to 12. RAPA binds to what?

They showed that it binds to at least 12 and 51. Okay, I mean, that's amazing. I had always thought that Rapa binds only to FKBP12, which then binds the tour. I didn't even realize it was binding to 51. So we know it binds to 51 in their paper and there have been some other papers studying the Ryanadine receptor that show that it binds to 12.6 also, which is in cardiac myocytes.

We've talked all about inhibition. Are there any times when you want to be activating this? There's a lot of talk that ketamine may be activating mTOR, and obviously ketamine has some really interesting properties as it pertains to recalcitrant depression. Yeah, so two points here. For patients with major depression, intravenous ketamine is almost a miracle drug. We're accustomed to typical antidepressant drugs requiring weeks and weeks to work and having modest effects at best.

Or even days, but this is minutes. Ketamine works in minutes to hours and a huge effect size. It's really amazing. I don't think we know what the specific cellular mechanism is of that. I'm giving you lots of things we don't know in our discussion. Wasn't there a study that showed rapa mice and blocked the effective ketamine? Yes, and there's a biotech company called Navator. I know them well, and they're using an intoractivator to treat depression.

Their Lloyd equivalent is also a very smart guy. I am. George is fantastic. Lloyd, is this a relatively recent understanding then about how Rapa is binding to the FKs and how it's, yes, the complexity around, first of all, how many of these things there are and how you can change their properties by which ones you're binding to? Yes. It's something that's not discussed a lot in the literature, but there are several FKBPs, where FK5 is six binding proteins.

We almost always talk about FKBP12, and it's sort of a shorthand, but the understanding has been that rapamycin binds to all of them in a few specific cases that's been shown to be true. There's also a bunch of other activities for enrolls for FKBPs that aren't at all part of torque one biology. For example, they all have an enzymatic activity. They're peptidyl prolylysomerasis, but what the biology of that is remains pretty much unclear. It's a very interesting enzymatic activity. It's involved in protein folding. But there have been some studies where maybe a dozen different of these immunophilons

are completely eliminated at the same time from cells. There was no clear cellular phenotype. So whether that means the others can all substitute because it's such a critical function or they have no function that's important, are there disease states where people are lacking any of these? There may be, but I don't know them. And how conserved are they across species? Highly conserved.

These immunofillons are present in almost all species, although they can vary a little bit. Is there any cell in the body that does not contain mTOR? I would bet some of the terminally differentiated a nucleot cells may not. Red blood cells, for example, do. Certainly, red blood cell precursors do. I was thinking about platelets, for example. I don't know if they have mTOR.

Yeah, interesting. And I bet that's known. It's just, I know the answer. And Itore. Yeah. Interesting. And I bet that's known. It's just, I don't know the answer. And I feel better that you don't. So we need to make a list of David Sapatini questions. Well, I'll make sure David listens to this. And you know what? Maybe we'll do an AMA with David specifically on tour.

So last thing I want to chat about because the paper came out kind of recently was this sort of interesting paper. It's interesting not in the sense of the intervention because it was an end of nine and it was a very poorly controlled study in the sense that there was no placebo group and every patient actually was on their own sort of tailored cocktail of three different drugs or two hormones and a drug. But the paper did get a lot of press because it used an epigenetic clock. Are you familiar with these clocks? Yes, this is the Horvaths work, right? Yeah, yeah. Horvath's probably the best of these clocks. Maybe it's just a little bonus episode. Tell folks how these things work, what they're measuring. We've already talked

a little bit about methylation. So maybe we put a bow on this by discussing that. So I forget how many years ago it was now, but it wasn't that many that we learned from Horevath and others that by looking at the methylation pattern on peripheral blood leukocyte DNA, we can tell how old you are within about six months to a year. And this has been replicated by several groups. So we're all familiar with DNA. And that's even true among centenarians and people that are just genetically blessed to live longer.

Excellent question. The studies that I've seen and actually the one that we participated in, I don't think we had people over 80. Okay. I don't know the answer to that. But for people between about 20 and 80, there's a stereotypical change in methylation patterns on DNA. And this is just a chemical change to DNA that happens over time, that's quite characteristic of your chronologic age.

This is what David Sinclair talks about as sort of the scratching of the CD. The CD being the master copy of your genomic. I guess we have to say something like that because we can't use wearing out of the vinyl anymore. But I think about it a little differently. I think about aging fundamentally as a biologic process controlled by pathways. And presumably it's a consequence of changes in gene expression and this methylation changes gene expression. So it's a pretty cool story and certainly we know that if you take a differentiated cell and treat it with a set of transcription factors called Yamannaka factors, you can reset the cell back to a pleuropotin stem cell,

and the methylation goes away, too. So, I'm thinking that DNA methylation likely could be causally related to the gene expression changes that not only are associated with aging, but make cause aging. So has anybody looked at the effect on the Horevath clock or DNA methylation in response to the rapologues we've been discussing today? I'm thinking of one experiment but I don't know that is published yet and I think the answer was negative. So it's interesting because the paper that I was talking about, again, there are 10 ways to Sunday this could just be an outlier, especially without a control group.

I mean, it's really difficult to make any conclusion. But if any of this benefit was real, which was growth hormone DHA and metformin, set the whorevath clock back, I think it was a year and a half or two and a half years. The initial hypothesis of the experiment was that this was going to improve thymic function, which was going to improve immune function. It doesn't seem like a stretch that you could potentially see that benefit from a rapologue if it's also acting on immunity, which is why I think I was sort of probably why I asked about the thymus in the past. Yeah, so I have a few comments about this. One is first DHEA we know goes down substantially with age, but there have been several to many studies of replacing it and there's no clinical benefit.

And I think the author used it for the effect of reducing the hyperinsulinemia that follows the administration of growth hormone. And in his, I think, personal experience taking growth hormone noted that he could blunt the hyperinsulinemia by taking something like 50 milligrams of DHEA by itself. But again, that's not something that's well documented in the literature. And your point, of course, is documented, which is DHEA by itself. But again, that's not something that's well documented in the literature. And your point, of course, is documented, which is DHE by itself. You can fix the number, but that doesn't seem to have any clinical bearing. Exactly. My second point is the growth hormone, the biologic activity of it is mostly driven, not exclusively by IGF1, which used to be called, I think, Somatamiden when we were in medical school.

We know that lymphoid tissue is probably the most sensitive target organ for IGF1 and it causes hypertrophy. So if he's looking for enlarged thymus in patients he's treating with growth hormone, I would be surprised if you did not see that. And he did. And so the question is, wouldn't you have expected that to have sped up growth? I would have expected, if he treated long enough, I would have expected to increase the size of the thymus, if he could find it in people. I believe the study looked at MRI and showed an increase in thymic size.

They were treated for a year, I believe. Not surprised at all. I bet the spleen increased too. Is there any reason to believe that that would enhance immune function? I'm thinking if I've read a paper about that and I don't know. And then what about the metformin wrench in the works? Metformins are really interesting compound. I think we have excellent data that in diabetics it is a wonderful drug. And there's some retrospective data of longevity in diabetics treated for a long time with metformin. And of course

this is the question near bars that I want to answer with the team study. Right. And near and I have spoken about this many times, and I agree that it's hard to make the case for a more beneficial agent in someone with diabetes or hyperinslamemia, metabolic syndrome or anything on that continuum and on that spectrum. Of course, the question is, what is the benefit of metformin in a perfectly healthy person or even a fully optimized person with respect to other variables. I'm not aware that there is a benefit of it.

Remember, it has some adverse effects on mitochondrial function too potentially. It's so funny you bring that up. That's exactly the question that I think most plagues me, which is if it is a weak mitochondrial toxin, is any benefit that you might see in a non-diabetic more than dwarfed by that downside, whereas even the simplest benefit of it, like a reduction in hepatic glucose output in diabetics, might more than make up for the inhibition of the mitochondria. I don't think we know the answer to that question.

It's one I'm super interested in and working on, and I'm actually gonna volunteer for a study that will take some muscle biopsies and look at peak mitochondrial function, which you can induce through certain types of exercise within without metformin. Interesting. Well, Lloyd, this has been just a fantastic discussion. I am so grateful for the introduction that Tim made. And it's been an honor to sit and speak with you.

I had tried to reach out to Joan about a year ago. I never heard back, so I'm gathering. It was just too busy at time. But in many ways, it was better to get to talk now because so much more has happened in the last year. And maybe that might have been just after the IPO. So it was a very busy time. I'm guessing my email went to spam.

But this has worked out really well. And I'm incredibly grateful for this. I wish you all the continued success with this program. Well, thanks. We remain optimistic and we will have top-line data from the first phase three by the beginning of 2020. So the data will speak for themselves. We'll count down the days till we see it. It's been a great discussion. I've enjoyed being here. I feel like I've said I don't know an awful lot, I'm feeling a little bit like I'm back in school, but it's been fun and I have some homework to do, thanks. Well, I think that's one of the beautiful things about folks that come on this podcast is great scientists saying I don't know, probably more than they know the answer, so that's I think a testament to your honesty. But thank you, like, appreciate it.

Thank you. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss, we've created a membership program that allows us to bring you more in-depth, exclusive content without relying on paid ads. It's our goal to ensure members get back much more than the price of the subscription. Now, to that end, membership benefits include a bunch of things. One, totally kick-ass comprehensive podcast show notes that detail every topic paper, person, thing we discuss on each episode. The word on the street is, nobody's show notes rival these. Monthly AMA episodes are asking me anything episodes, hearing these episodes completely.

Access to our private podcast feed that allows you to hear everything without having to listen to spills like this. The Qualies, which are a super short podcast, typically less than five minutes that we release every Tuesday through Friday, highlighting the best questions, topics, and tactics discussed on previous episodes of the drive. This is a great way to catch up on previous episodes without having to go back and necessarily listen to everyone. Steep discounts on products that I believe in, but for which I'm not getting paid to indoors, and a whole bunch of other benefits that we continue to trickle in as time goes on. If you want to learn more and access these member-only benefits, you can head over to peteratiamd.com forward slash subscribe.

You can find me on Twitter, Instagram, Facebook, all with the ID, Peter, Attia, MD. You can also leave us a review on Apple Podcasts or whatever podcast player you listen on. This podcast is for general informational purposes only. It does not constitute the practice of medicine, nursing, or other professional healthcare services, including the giving of medical advice. No doctor-patient relationship is formed. The use of this information and the materials linked to this podcast is at the user's own risk.

The content on this podcast is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Users should not disregard or delay in obtaining medical advice from any medical condition they have, and they should seek the assistance of their healthcare professionals for any such conditions. Finally, I take conflicts of interest very seriously. For all of my disclosures in the companies I invest in or adviseto-date and active list of such companies. you