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

Hey everyone. Welcome to the Drive Podcast. I'm your host, Peter Attia. This podcast, my website and my weekly newsletter all 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 peterattiamd.com subscribe now. Without further delay, here's today's episode. I guess this week is Lloyd Klichstein. Lloyd's the Chief Scientific Officer at Restore Bio. So that's Little Lloyd r e s big T o r little B I o get it Tor t o r Restore Bio is a clinical stage biopharm company that develops meds that are primarily aimed at targeting tor target of rapamycin. 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 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 rapalogs, and we talk a lot about one of them in particular called Everolimus. 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. A December 2014 paper. Joan Manik 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 mention, of course, that Lloyd is an employee of RestoreBio. Restore Bio is a for profit company that is working on MTOR inhibition. 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. Now in the interview we discussed an upcoming phase three trial from RestoreBio. 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 Manic along with Nir Barzilai. I'm going to have the two of them back on an episode where we're going to discuss a whole bunch of things that'll be quite interest. 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 Manik 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 were 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 Klickstein. 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's 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 of that effect. You're 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 meaning I was so excited when we sat there. And it was. So it was with Tim and with DA Wallach, 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 always refer to it 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 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 Kaeberlin, 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 Rappa 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 be basically physician, scientist, and then ultimately now move into 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, or both.

So did you do a combined MD, PhD 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 challenges, and 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 a 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 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 and I'm constantly amazed. In fact, I was interviewing someone recently, a very remarkable scientist, an 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, is the point.

No, and it was much more challenging than I had seen it in the late 70s and early 80s.

What changed is the reduction in grant. The competitiveness of the grant environment or.

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. Novartis Institutes was created by Mark Fishman 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 has 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 have a dedicated podcast 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. Jennifer Doudna 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 would it work in humans. Who would have thought that would work? Bacterial chromatin, the DNA is so different. It's supercoiled in a bacterial cell, whereas human DNA is 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 antihypertensives 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.

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. It's 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. 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 Novartis in 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 Novartis was working on. It was a regenerative medicine concept in 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 a drug called zoledronic acid to increase bone 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 is, I suppose, is. Do you want to tell folks what that is?

Sure. So an IND 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 is they separated research from clinical development. Other companies did 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 in 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 Fishman, who was 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 9. We basically applied those principles. A real medical need, a problem that's scientifically tractable and that Novartis wasn't working on. And ideally that nobody was working on. And we ended up with a few very interesting areas. Joan Manock, 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?

Now, was Joan at Novartis at this time? Yes.

She joined our group in part to do this. Her real innovation, beyond just the idea, is 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 timeframe, 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, as in, 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 Joan 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 Kaeberlin. 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, at the risk of oversimplifying, grow or don't grow.

Yes, I think that's exactly right. The signals it 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 cell should grow or not.

And between. This is just a little history lesson for the listener, sort of between 1991, when hall first identified what was not called at the time TOR, but what would go on to become TOR 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 conserved 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 lysosome, 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 can sort of picture this endocytotic process as the cell membrane or wall, depending on what if it's eukaryotic or prokaryotic, 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 vacuole 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 Metchnikov 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 watch them fight bacterial infections. And he was a major champion of something called cellular immunity. At the same time, some German scientists, notably Paul Ehrlich, were working on what we now understand as antibodies. And they said, no, it's humoral immunity or soluble immunity in your blood. And we 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 MTOR is probably on a lysosomal vacuole, because in the context of evolutionary development, it was on these vacuoles that very simplest organisms use 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 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'd turf that question to David Sabatini.

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 mtor, which again, it's one of those things that's funny when you start to explain it to people, because you can't explain what MTOR is without somewhere explaining what rapamycin is. Given the name MTOR stands for mechanistic target of rapamycin. But David also played the fundamental role in elucidating that MTOR can be organized in a couple of different ways and sort of 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 TOR proteins, 1 and 2, 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 Torc1 for target of rapamycin, complex 1 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 cell's 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 its 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 immunophilin called FK binding proteins, or fkbp. And there's several of these. There's three different classes of immunophilins. The complex of rapamycin bound to FKBP then binds to the torque one 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 phospho S6 kinase, which name's 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 4ebp1, and even less so for a target called ulk1, which is involved in activating the cell's 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 it binds pretty tightly to this binding protein. This binding protein then moves towards tor, and in the case of. Did we explain Raptor and Richter yet? We haven't explained those case. Do you want to spend maybe just a minute so that they can see the difference between complex 1 and complex 2?

So MTOR is present in both torque 1 and torque 2 complex, but there are proteins that are unique to each complex.

So as you were saying, the yeast have two different Tor, everything else has the same Tor, 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 mtor. 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 1 complex, and Richter is unique to the torque 2 complex.

And they're covalently bound to Tor.

I don't think it's covalent.

Okay, 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 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 rapamycin FKBP to torque one.

Yes.

And then that alters the torque one 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. Rapamycin 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 1 does other things too, particularly in terms of regulation of lipid synthesis, pyrimidine synthesis. Pyrimidines are part of DNA.

What about MTORC2? So how does Rapa and FKBP bind to MTOR Complex 2?

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 resynthesize enough tor? Is there a shortfall of torque because so much of the TOR is bound to the RAPA FKBP complex that you now run out of enough tor to make MTOR complex 2?

You could imagine that as one possibility, but I don't think that's the case. I think it's more. There's a feedback signaling pathway that down regulates torque 2.

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 rapalog 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 2 inhibition with Arapolog alone. And it's reflected in hyperglycemia and hypertriglyceridemia. So biomarkers in the peripheral blood you can measure.

Why is it that inhibition of MTORC2 leads to 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 immunocompromised, quote, unquote, as normal as possible subjects, do we have evidence that those things happen?

We do. MTOR inhibitors. Well, rapalog specifically have been tested in non transplant, non malignant disease patients. Some specific examples include the RAPALOG RAD001 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 could 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 5mg if it wasn't tolerated.

Is RAD001 identical to rapamycin in dosing? 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 MTOR complex is a little different. But if your rapamycin dose is somewhere between 2 and 8 milligrams a day, roughly at the immunosuppression level of dosing, which is what we're talking about, that's comparable to 5 to 10 milligrams of RAD001.

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 2 seems to produce things that are not really desirable at all, whereas the output of an MTOR complex one inhibition pathway seems quite desirable, at least sometime?

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 I, but you still have MTOR and Richter, so therefore you have complex two, what does that animal look like?

Depends on how you do it. If you imagine making a mouse that doesn't have raptor one, which means it doesn't have TOR complex one from conception.

I assume it has muscular dystrophy or something like that.

They're embryonic. Lethal. Yeah.

Okay.

You need Torque 1 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 or knock out torque one.

So once they're out of the development window and they've reached adult size, then it's better tolerated. Yeah.

And again, David Sabatini 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 1 complex. Inhibition of torque 1 extends lifespan and health span in rodents, if you do that to torque 2, it accelerates death.

Such an interesting concept. I mean, because what it basically suggests is, at least with the tool that we currently have to block tor, which is like rapamycin or rapalog, giving intermittent dosing may be 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 rapalog with continuous administration, yes, you'll eventually downregulate torque 2 as well. And as far as we know, that's not favorable. There are some other ways to do this, And Joan Manik'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 rapamycin 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 sirolimus 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 Listeners, 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 arapolog could alter the biology of lymphocytes.

And you're saying that in a favorable way, not a disfavorable way? Yes, because the earliest observations of Suren Sehgal 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 paper.

Yeah, yeah, exactly. Like lower or higher, I guess, is what I'm saying.

Relative to that, it's hard to compare as you have to look at exposures. So the doses on a weight basis or a body surface, they're much higher. Were 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 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 listeners, 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 it's just. I was talking to a patient about this the other day. Cialis is typically given as either 5mg or 20mg, 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 erectile dysfunction is ameliorated Friday through Sunday. Conversely, another way to take Cialis is to take 5 milligrams every, 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 Cialis taken once might be in your body for a day. A 20 milligram dose of Cialis 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 Cialis is long enough that it can do that. That is not the case for Viagra, for example.

That's right. Now, there's another interesting thing here, which will also be another will have a parallel to the Rapa story, 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 phosphodiesterase inhibitors.

Yeah, the 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.

We measured, yeah. Daily. I mean, we were constantly doing this.

But you measured the trough levels, not the peak levels.

Not peak, 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 gentamicin and a lot of the negative, the antibiotics that have the same toxicities on the trough, Right?

Yes. So gentamicin, years ago we used to dose three times a day. 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 gentamicin or rapamycin is producing toxicity by its nadir, not its peak?

Every drug is different and it depends on the specific mechanism for gentamicin and aminoglycosides 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. 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 let's now taking that model back. The first glimmer of hope that this drug had wasn't uniformly immune suppressing was well, 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, how rapamycin bound to the two different complexes to test this hypothesis proactively or was this more empirical an 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 calcineurin inhibitor versus those who were on arapilog. And there was a significant trend that those on rapalogs had fewer cytomegalovirus infections than those on calcineurin 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 cyclosporine.

Yeah, yeah. Well it was rapamycin versus cyclosporine 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 Torque 1, 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 arapolog and test whether their immune function was better, we couldn't be giving them a typical arapolog 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 1. 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 rapamycin and rapalog. So again, a little bit of a history lesson, but rapamycin is the name given by Suren Sehgal 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 Sirolimus, and that was FDA approved in 1999 for transplantation. What was the first Rapa log to come along?

Arguably it's Rapamycin, the next drug.

Yeah, sorry, Semantics aside. Yeah, yeah. After Rapamune. Rapamycin. Cos 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 RAD001 in the paper. The generic name is Everolimus. It was the second one Tem. Sirolimus is another one.

How does it differ from Rapamune or Sirolimus?

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 Rapamune 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 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, it was embargoed for another couple of hours. The person at the Times who 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 experiment 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, Joan 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, 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 1's biology, while in the big picture, is 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 1, yet give less than assay trough levels to help ensure safety in 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 I 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 MTORC1 inhibitor?

You're asking really for Novartis, thinking that's probably still confidential. But the big picture is we want to know if something works. We want to figure out if we can make it better again. Ultimately, Novartis, we at RestoreBio, 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, RAD001 was FDA approved for another indication or no? Yes, it was already approved in cancer or in.

I think it was in renal cell rcc. Yeah.

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 suspect most companies do things like this where when the 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 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 Novartis had 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 all in 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 about to get their flu vaccines. In that paper, we were ready for the Southern hemisphere because our summer is there. Winter.

Right. And since the CVS in the Antarctica ran out of vaccine that year, Australia made the most sense.

Australia and New Zealand.

Yeah. So there are four arms in this study. There's a placebo arm that gets just obviously a placebo. There's three treatment arms, one that gets 0.5 milligrams of RAD.001 daily. So everolimus, 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 5 and 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 0.5 daily versus the 5 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 is 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, and there were several, 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 1 inhibition. The 0.5 milligram dose partially inhibited in a sustained fashion. The 5 milligram once a week fully inhibited torque 1 for a couple days out of the week. And the 20mg we modeled would fully inhibit torque 1 over the dosing interval.

I didn't realize that actually 20 is so high that it gave you functionally non stop inhibition of MTORC1 until your next dose. Did any of these have MTORC2 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 1 is inclusion criteria or something like that, but you guys just skipped the foreplay and went right to it. Table 1. Incidence of treatment related adverse effects I wish I could honestly say I remember how I read this the very first time. Cause I've looked at it 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 or 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 ARM1, it's 22, in ARM2 it's 20, in ARM3 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 aphthouse ulcers with their daily dose of rapamycin. And most of them were getting more than point daily. So most of the patients that I my recollection was that 2-4mg was a very common daily dose for Rapamune. And again, this is Rad.001, 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 eleven and a half percent of these folks had mouth ulcerations, whereas the people getting 0.5 daily were at 0.5 once a week was about 4% and 20 once a 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 this study?

Well, as you pointed out, mouth ulcers are one of the more common and fairly specific side effects for rapalogs.

Should we spend one moment explaining why? I think we could certainly speculate why.

I don't think anybody knows why. There are some hypotheses, but. And it isn't just rapalog associated mouth ulcers. We don't know what causes the ordinary spontaneous apthys ulcerations that people get. There's 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.

Anecdotally, through five years of residency, I don't think I went more than two weeks without an apathys ulcer.

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 bupivacaine 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 bupivacaine 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 bupivacaine in and I'm kidding. They must have thought I had some drug problem. But two weeks after leaving residency, I never had an apathys ulcer again.

I hope you never have another one. They're very painful.

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.

Yeah. 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 a placebo. The study was randomized, double blind, and placebo controlled. So neither the doctors nor the patients knew 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 something.

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. In the high dose, we just saw.

One strain, one of the three strains.

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 arapolog are immunosuppressive. The dose of 20mg once a week is sufficiently high to fully suppress torque 1. And 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 or an upside down J curve, really? Or 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 lymphoproliferation? 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 an antibody titer assay.

Right.

We didn't do mixed lymphocyte reactions or some proliferation assay as 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 lymphocyte assays. Part of the study was a very comprehensive multidimensional flow cytometric assay to get lymphocyte subsets. And we reported one of the results in Figure 3, 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 Keytruda or Opdiva.

We had Keith Flaherty 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 rediscussing 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 lymphocytes 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 a lymphocyte 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. There you go. That's how I do things. So has anyone looked at this, by the way, to see what rapamycin does or rapilog does in this type of intermittent dosing to inhibitory T cells?

There have been a lot of studies of T cell subset functions with rapalogs, both in mice and in humans. Looking at effector memory transition, looking at Tregs with high exposures, there's substantial inhibitory effect on B cell function. There's a lot out there.

Do you think that rapalogs could be used to suppress Tregs selectively? Of course.

There are a couple of papers that.

Show that, because it would sure be interesting to start layering in rapalogs with immunotherapy, oncology specifically? Yes.

Yeah, there's 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 B cell, 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 rapalog we used augmented autophagy and could that contribute to antigen presentation? We saw and we measured seven of the 23 antigens.

Wait, tell me why that would be. That's not an obvious purview into autophagy 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 1.

This isn't a peptide presentation. This is presented in the context of an innate 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 lipopolysaccharide and then.

And directly stimulate 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 here for autophagy? I mean, 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's paper on unpaired hemoglobins 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 banked 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? I mean, how much of the benefit here is through direct inhibition of mtor? And are there any pleiotrophic benefits that aren't quantified through this? Obviously some of them. I mean, 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 upregulates 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, of a Rapalog? I think the mouse experiments tells us there is. And it's because relatively short course of a Rapalog 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 Jay 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, 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 Rapalogs and it doesn't seem to happen.

In other words, you take a Horvath clock pre and post 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.

Yeah. So we're high fiving on New Year's Eve 2014. How do you go from what is Novartis again? If this is confidential, by all means, just gloss. You know, we'll skip it. But what do the brass at Novartis think of the results of this experiment, which is effectively taking a drug that we already have on the market from. 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. A lot 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 a 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 1000 patients, the other one will have about 1600 patients.

You able to speak about how the decision was made for restorebio to basically acquire a piece of an asset from Novartis and what else was brought in to create 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 a vested interest in the success of restorebio.

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, 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 one 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 internal. Any clinical study like This, a phase two study with two to 300 patients, costs millions. And 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 1 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, and this is published by Beat Neifeller and Lone Murphy. They showed synergy.

So, Arapologue, can you explain to folks what the difference is between alosteric versus catalytic inhibition?

Sure. So catalytic inhibition means an inhibitor that's binding at the catalytic site of Torque 1 and blocking phosphorylation of targets. An allosteric inhibitor is a fancy way of saying an inhibitor that binds someplace else on the molecule and nonetheless inhibits it. Rapamycin complements.

I always think of allosteric as sort of shape blocking.

Could be, yeah, let's say shape blocking. So in this case, the allosteric inhibitor is the combination of FKBP12 plus rapamycin binding to torque 1. So the combination of those two synergistically inhibits torque 1 and it's a little more torque 1 selective.

Everolimus does the same thing. It also binds to fkbp.

Yes, it binds to fkbps also. Maybe later on we can get to that lambing 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 RAD001. 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 few 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?

Too 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 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 Godonkin, right?

Yes, I agreed. 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 listings.

So what were some of those infections, Lloyd, that you saw? 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 metapneumovirus, there's various other echo, God only knows what. 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 sometime 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 revaccinated 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 4 plus 2 or 3 plus 2 or, or 2 plus 2? Would the benefit have been better if you went 8 plus 2 or 10 plus 2 or 12 plus 2. 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 vaccinated, eight weeks, have some new lymphocytes from those committed precursors.

So your Hypothesis would be 6 was the minimum time required to get a full turnover. And going 8 versus 10 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 rapalog, 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 endpoints. So we're looking at them prospectively, were collecting them more carefully.

That was in the second study, though.

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 was a new molecule?

Yes, it's a new compound. It was in that paper. It was called BEZ235. And this is the molecule licensed by RestoreBio.

So BEZ235 is not RAD001 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. This 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, A year ago. 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 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 in drug development. You never know exactly what's going to work best. You 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 and this fell below the funding line. It reflects in part the excellent productivity of Novartis 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, 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 of Novartis 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 Restore Bio.

And that was a pretty quick path to going public. RestoreBio went public late in 18, didn't they?

It reflected in part the need for funds to run a phase three program.

Yeah. What did RestoreBio 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, you're basically going right to phase three at this point because. So you licensed one or two molecules from Novartis.

RestoreBio licensed BEZ235, which is now named RTB101, and they then did a phase IIb study. These two studies we had done were phase 2a.

Phase 2a.

Yeah. Right. 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 a priori, 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 RTB101, 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 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 BEZ235 or RTB101 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 I, as it is binding to Tor, bound to Richter, known as Complex 2.

So the way this is usually done 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 1 and Phosphoakt there. And we look specifically at the phosphorylation site that Torc2 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. RTB101 has some preference for Torque 1, 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 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.

Wands, no magic wands 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 prodrug 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 are you 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. 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 RTB101 was basically the basis upon which Restore Bio was formed, Correct?

That's right.

Now, I was very confused. During your roadshow, which was about a year ago, maybe more than a year ago, it might have been early 2018. I've certain sort of lost track. You were at Novartis at the time, so it wasn't really your roadshow at the time. But I naively, I guess, thought that RTB101 was actually RAD001, the everolimus 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 Everolimus combined with a PI3K inhibitor. So how were we confused by that?

When RestoreBio was formed, it licensed RTB101 from Novartis for all uses. And there was also a limited license to use RAD001 only in combination with RTB101 for our indication. Now, the Phase 2B study that RestoreBio ran showed that the most effective drug or drug combination for preventing respiratory tract infections was just RTB101 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 RTB101 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 MTOR 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 MTOR in order to express 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, two once a year is probably not adequate. So there's some frequency upon which you want to metacycle that. But the reason you don't just want to go all the time taking it, presumably, is you might actually start to impair protein synthesis. That's necessary to, 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 rapalog. Mitch Weiss's 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 arapolog. So I think there are some proteins that are sensitive to translational inhibition by Rapalogs and perhaps some that are less sensitive.

Before we go down this path of getting a little bit more into RTB101, I want to 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 Kaeberlin is seeing in dogs, if you think of sort of the global excitement that exists around rapamycin and rapalogs, 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 autophagy, targeting of and or destruction and or desilencing of senescent cells, reduction of inflammation, enhanced mitophagy, what other mechanisms do you think could be involved here and what evidence exists to support or refute that?

Well, I think we know from academic experiments that every single one of those mechanisms can extend health. Spanish 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 Sengupta, was looking at the consequences of fasting on MTOR activity in young people. As you would expect, fasting leads to suppression of MTOR 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 in old mice that's impaired. We've only done the liver tests in mice. So we back translated this experiment and gave. Actually, I think it was rats. It was old 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 MTOR 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. They'd already passed that stage where caloric restriction would extend their life. And yet their lives were extended 15 and 25% by rapamycin. 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 knew 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 MTOR 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 S6 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 intervention conventional 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 every one that.

Yeah, there's sort of a referral bias. You never see the thousand that go, well, yeah, you only see the one that didn't.

Yeah. 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 autophagy is impaired in older folks with fasting? Because autophagy 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 autophagy. There's other mechanisms. There's beclan one 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 fishing 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 RTB101? 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 two A study that Novartis ran, we saw decreased respiratory tract infections in elderly people treated with it. In the phase 2b study that Restore Bio ran 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 RTB101 plus RAD001 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 Novartis 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 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. Yeah.

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, 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 in. Involved in organ transplantation today is remarkable.

A subject for another time.

I know. And all started at your alma mater. 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 3 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 IIB 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 5 milligrams and 10 milligrams. We did a different schedule, we did 10 twice a day. And we did a combination with a RAPALOG, our RAD001 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 MTOR partially for part of the day.

By the way, if you had to speculate going back to the very, very first, the 2A with RAD0001, if you had taken all four of those groups and measured them at the end of six weeks and then after the two week washout, what's your prediction as to how they would have differed?

I'm sorry? Cause we didn't vaccinate until if we vaccinated at six weeks versus vaccinated at eight 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 washout. 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'll bet we wouldn't have.

And do you define high as 5 and 20?

High as 20, yeah.

Okay, got it. So you think the 5 and the 0.5 still would have had benefit on drug? 20 probably got a 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 of 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 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 MTOR 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 sub studies in this 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. There 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 diluted. I'm just saying you, 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 more consistent. 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.

Yet we saw consistency.

Yeah. So I think we're saying the same thing, which is, it's more a credit to the finding. 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 a startup to take.

And so now we've seen 10 milligrams of RTB101 decrease respiratory tract infections in the Phase 2a and in each of the parts of the Phase IIb. And we use the Phase IIb 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 that we're calling the program, the Protector program, the first one is fully enrolled, 10, 24 patients. They're getting 16 weeks of drug treatment and we're following them for respiratory tract infections with sort of 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 10mg 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 Lemming paper that came out about two months ago. That's a prolific lab. Lemming was a postdoc in David's lab, so no stranger to this science, there are lots of folks out there that are working on selective MTORC1 inhibition notwithstanding, the potential Ways around it through intermittent dosing or looking at catalytic binding or things that might be offer 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 laming group looked at, I think it was 90 different rapalogs presumably related to rapamycin and looked for their ability to be selective for Torque 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 immunophilin discussion, is that they found possibly the reason the compound was selective was that it bound to one of the immunophilins but not another. So specifically it bound to FKBP12 or at least FKBP12 was required for the compound to have activity, but another FKBP, FKBP 51 was not.

But I still don't understand this. If you bind to fkbp12 and then the rapalog 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 rapamycin 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 serine kinase or the 4ebp one or something like that, like something downstream of a direct phosphorylation is counter regulating or. Yes, yeah, I see. And you're saying presumably if you only bind arapilog 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 2 activity at all, does not bind to FKBP51 or at least that's the implication because down regulating FKBP51, which I think they did with an SIRNA, had no effect on its inhibitory activity, suggests that there's a complexity to the complexes for formed. Sorry for that that we don't yet understand and it's an exciting area to explore. So remember, every cell has many immunophilins in it. It has several cyclophilins, 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 FKA BP 12, which then binds the Tor. 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 ryanidine 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 can 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 rapamycin blocked the effect of ketamine?

Yes, and there's a biotech company called Navitor. I know them well, and they're using an MTOR activator to treat depression.

Their Lloyd equivalent is also a very smart guy. So George is.

Yes, George is fantastic.

So, Lloyd, is this a relatively recent understanding then, about how Rappa is binding to the fks and how it's 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.

Yeah, so it's something that's not discussed a lot in the literature, but there are several FKbps or FK506 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 and roles for FKBPs that aren't at all part of Torque 1 biology. For example, they all have an enzymatic activity, their peptidyl, prolyl, isomerasis. 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 immunophilins 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 immunophilins 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 anucleot 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 I feel better that you don't.

So now 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 Tor. 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 n of 9 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. Yeah. This is Horvath's work, right?

Yeah, yeah, Horvath's probably the best of these clocks. Maybe just as 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 Horvath 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 the. What David Sinclair talks about as sort of the scratching of the cd, the CD being the master copy of your genomic.

Yeah, 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 Yamanaka factors, you can reset the cell back to a pluripotent 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 may cause aging.

So has anybody looked at the effect on the Horvath clock or DNA methylation in response to the Rapalogs we've been discussing today?

I'm thinking of one experiment, but I don't know that it's 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, DHEA and metformin, set the Horvath 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. Doesn't seem like a stretch that you could potentially see that benefit from a Rapalog if it's also acting on immunity, which is why I think I was sort of. That's 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. 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, Somatome. Eden, 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?

Metformin is a 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 Nir Barzilai wants to answer with the TAME study.

Right. And Nir 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 hyperinsulinemia, metabolic syndrome, or anything on that continuum 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 than make up for the sort of inhibition of mitochondrial.

Yeah, I don't think we know the answer to that question.

It's one I'm super interested in and working on and actually going to 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 with and without metformin.

Interesting.

Yeah. 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. You know, 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 3 by the beginning of 2020, so the data will speak for themselves.

Well, 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 say I don't know probably more than they know the answer. So that's, I think, a testament to your honesty. But thank you, Lloyd. Appreciate it.

Thank you.

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