So, having mentioned that important disclaimer, in general terms, can you highlight the differences for their prescription of rapamycin for their original purpose, you know, for the organ transplant and anti-aging or other indications, including Alzheimer's disease for the nontransplant patients? Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. What are the differences in frequency and dosages in observed or expected side effects just in general terms again? Not giving a piece of medical advice is important to mention once again.

Sure, yeah. So So in organ transplant patients, you know, there's a bit of variation in the typical regimen, but this is my understanding, I'm not an organ transplant doc. So this is based on what I've read. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And from talking to organ transplant Doc's, but my understanding is typical, you know, I think rapamycin, which is called sirolimus, in the clinical community, was first approved for kidney transplant rejection. So that's probably where there's the most data. And usually there's what's called a loading dose, which is a higher dose, you know, maybe four to 10 milligrams a day when patients first go on rapamycin, and then there's a maintenance dose, which is typically in a couple of Meg's a day, so it's a daily dosing, oral, usually tablet. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And that's dose based on, you know, desired peak and trough levels in the blood. So there's a little bit of individual dosing that goes on there. Still, it's typically in the, you know, a couple of milligrams, maybe even a little bit higher, sometimes everyday paradigm, and it's continuous, right. So, you know, I think if you've had an organ transplant, the reason for taking immunosuppressants and sometimes rapamycin or other mTOR inhibitors is to prevent rejection of the transplanted organ. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. You take your medication every day to prevent rejection of the transplanted organ, and it's usually continuous for the rest of your life. Now, organ transplant patients sometimes come on or go off of different medications, including rapamycin. Still, you're going to be on that immunosuppressive regimen, you know, for the rest of your life. So that's very different than the context of potentially using rapamycin for health maintenance for disease prevention, which is the way I think about it. I think most people, you know, who are studying rapamycin in this context, think about it. It's really to keep people healthy. It's not to treat a disease. And so, in that context, the first thing I think it's important to appreciate is it's all guests. It's all guesswork, right? It's educated guesswork, and it's based on data. But there haven't been double-blind, placebo controlled clinical trials, really, to evaluate what is the best dosing paradigm for rapamycin in this context. So what most people have settled on is his once-weekly dosing. Again, oral tablets are usually in the four to six milligrams once a week. And there's some variation there. Some people do lower than that. Some people do higher than that, but that's kind of the ballpark typical rapamycin dosing regimen right now. And that's really based on anecdotal data from a number of people who are public about their rapamycin use. And from a couple of really nice, relatively good-sized, randomized, placebo-controlled clinical trials with a drug called ever alignments, or rad 001, which is a derivative of rapamycin, it's what's called a rap along it's just as a slight chemical modification on rapamycin that changes the bioavailability a little bit. And in those studies, in this dosing range, like five milligrams once a week, this was in healthy, older people. The side effects were essentially no different from placebo in the everolimus group with a couple of minor exceptions, and I'll mention that in a minute. And it appeared to have efficacy for immune function in the elderly, specifically influenza vaccine response, which is interesting and subsequent infection with several different viruses including Coronavirus, which is also interesting given the world that we live in today. But I think the take-home here is that it seemed as if the rapamycin derivative was at least partially restoring immune function in healthy older people that allowed them to mount a better vaccine in response to a flu vaccine and potentially protected them against other viral infections in the subsequent year. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. So that's the data that I think is really guided this development of a once-weekly dosing paradigm with rapamycin in that four to the six-milligram range. So what are the side effects? So in organ transplant patients, I can't, you know, it would take me half an hour. Well, that's, that's exaggerating. It would take a long time to read through the entire list of side effects that are in the box, right? For many of those, it's unclear whether they're even real side effects in organ transplant patients. And this gets to how, you know, manufacturers are required to list side effects on FDA labels, but some of the ones that seem to be pretty clearly caused by rapamycin in that context are hyperlipidemia. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. 

Increase in mouth sores, a somewhat increased risk of infection. That's what you would expect for organ transplant patients on immune suppressants, gastrointestinal effects, potential defects in wound healing. And then the other one that I think concerns people in the context of potential use as a preventative is an increased risk of something like a pseudo diabetic state in organ transplant patients where there is a decrease in glucose homeostasis and insulin resistance that's seen in people that take, you know, daily rapamycin for a long period of time. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. There are a few others, but I think those are the ones you know you would be most concerned about, particularly the increased risk of infection, for healthspan, promoting the use and the potential for metabolic defects in glucose homeostasis. So what has actually been seen, and again, it's important to appreciate there isn't much in the way of double-blind placebo-controlled data, especially for longer-term. So I think what we can say is over the short term, six to 10 weeks in humans, at once weekly rapamycin dosing, there's almost nothing in the way of significant side effects, maybe with the exception that some people experience mouth sores, and, you know, this is really like canker sores in the mouth. So not, you know, not life-threatening, but you know, maybe not particularly enjoyable. But beyond that, there's really no evidence for significant side effects. What is a little bit, and this is where there's just an absence of data. And we hope to help address this and the project that I mentioned, you know, when you get outside of that six to 10-week window, what is the real risk of side effects. And again, this is now just anecdotal. In my experience talking to people, the one thing that seems probably real is that there is maybe a two-fold increased risk of bacterial infection. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And again, that kind of makes sense with what we know about the effect of rapamycin on the immune system. So not much data to support that, but my intuition is that it is probably a real side effect. It's not a huge increase in risk. And, of course, treating bacterial infection is pretty easy with antibiotics. So it's, you know, as long as you are aware of the risk, it's manageable. The interesting thing, and you know, this comes from conversations, mostly with Alan green, is he very much believes that there is a sort of the corresponding benefit. And this has to do with the effect of rapamycin on the innate immune system versus the adaptive immune system that at the same time that you get a slight increase in the risk of bacterial infection, you actually get a pretty potent increase in resistance to viral infection, which fits well with the study that I mentioned with everolimus. So that is highly speculative, I would say, but something that many of us are interested in understanding further, and again, in the middle of a global viral pandemic, you can easily understand why, you know, that would be interesting and important to know, if something like rapamycin could actually have a beneficial effect it especially in the context of the age, the immune system on viral resistance, so I'm sort of, you know, interested to see how that data plays out over the next couple of years.

Well, that's thank you for this overview. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And it's very important to dive into the nuances of side effects, you know, a slight increase in bacterial infections, of course, it's important to note because, you know, it is older adults who need to be vaccinated against pneumococcus, there are specific vaccines that are recommended for older adults, but at the same time, they don't mount this significant response to bacterial pathogens. So if it's a two-fold increase in, you know, meningitis, that can be potentially fatal. That's, that's the square root of concern. On the other hand, if it's balanced by a decrease in the risks, for other diseases, that's also important because that's a significant worldwide discussion that people have on aspirin that people have on even alcohol, you know, there are certain risks that are decreased and certain risks that are increased and where to find that balance in those and frequency. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And I guess that applies to both pharmaceuticals.

And diets. Remember, as we talked about, right, diets have biological effects do so again, I think, you know, it's hard to know what the absolute risk-reward is, in any of these cases, because there's always going to be some lack of data, something like aspirin, certainly, you know, we know a lot more about the potential risks of aspirin. So you can, you can evaluate that equation, you know, maybe a little bit more precisely, with rapamycin. I think we just don't have the data yet. And with things like, you know, protein restriction, people don't even think about it, right? People don't even really think that there's probably a risk associated with protein restriction, particularly in the elderly. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. Like, I think that's a really interesting example. Because, you know, there are people in my field who study protein restriction in mice who are making recommendations to people that they should practice protein restriction that might be good in a young person. It's pretty clear, at least from my view, from the geriatric literature, that protein restriction in the elderly is probably not a great idea, right? But there are people who immediately extrapolate from mouse studies to humans and start making recommendations to humans and never even stop to consider that there may be unanticipated consequences to something like protein restriction. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. 

Well, that's, that's certainly true. And that's important because it's a function of the immune system and elderly people, the aging immune system. Dr. Matt Kaeberlein, PhD. Dr. Anton Titov, MD. And that's clear, you know, once you restrict the protein, that could be detrimental effects, as you mentioned.