Hey.

Everyone. Welcome to The Drive Podcast. I'm your host, Peter Atia. 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, and we've established a great team of analysts to make this happen. It is extremely important to me to provide all of this content without relying on paid ads. To do this, our work is made entirely possible by our members. And in return, we offer exclusive member-only content and benefits above and beyond what is available for free. If you want to take your knowledge of this space to the next level, it's our goal to ensure members get back much more than the price of the subscription. If you want to learn more about the benefits for our premium membership, head over to peter. Etia. Md. Com/subscribe. My guest this week is Colleen Cutcliffe. Colleen received her bachelor's degree in biochemistry from Wellesley College and a PhD in biochemistry and molecular biology from Johns Hopkins University. She then completed postdoctoral training at Northwestern Children's Memorial Hospital and subsequently began working as a scientist in the pharmaceutical industry.

She's currently the CEO and co-founder of pendulum Therapeutics, a startup that is working to develop treatments for a variety of diseases by targeting the microbiome. Although, given that pendulum has been around for about a decade, it seems a little odd to refer to them still as a startup. I wanted to have Colleen on the podcast because, quite frankly, she was the first person I met and had deep discussions about the microbiome where I really felt like the person knew what they were talking about. Now, I don't say that to be disparaging of the many other people who have a lot to say about the microbiome. But my mind works in a particular way, and I guess I've just never had that connection with a person where when I ask questions, they seem to have answers that make sense to me. So much of the work that I've seen around the microbiome has been interesting, but it's been very difficult for me to understand how one could operationalize and make real causal effect from the science that is being presented. And so over the course of many months, Colleen and I had a number of discussions.

I became quite interested in some of the products that her company sold, and I even began to see some of the benefits in my own blood work, something that I was incredibly skeptical of at the outset. In fact, we realized after our first meeting that many people had been telling me about some of these products over the previous four or five years, and I had been generally quite dismissive without looking more deeply at the data behind them. In this conversation, we really dive into all things related to the microbiome. We talk about what it is and how it changes over time and how you can measure it if that is something that is important, and we'll talk about the importance or lack thereof. We talk about probiotics versus prebiotics versus postbiotics and how much bacteria actually make it into various products that one can buy. We speak about fecal transplants and the importance of fiber as well as artificial sweeteners and antibiotics as it relates to the gut microbiome. We then speak specifically about Akkermansia and Colleen's work at pendulum around creating products that focus on not just Akkermansia, but creating substrate for it and other tools that will enable the gut to be fed in the best possible way to improve metabolic health.

This is a really interesting discussion on many levels. And again, I just want to stress how skeptical I have been of this topic for at least a decade. In fact, there is probably no area of medicine that I have been more skeptical of than this one. Of course, with some of the high-profile failures of companies in this space, I have somewhat smugly felt vindicated in my skepticism. But I must say that I feel like that is changing. I think that the work that Colleen and her team have shared with me, along with work that has been shared by other people, have began to make me think that there may indeed be something to this gut biome. In other words, it's not that I don't think that the gut biome matters, but the question is, can we change it using tools other than our nutrition? And to me, that's one of the most important questions we dive into in this discussion. So without further delay, please enjoy my conversation with Colleen Cutcliffe.

Hey, Colleen. Thank you so much for making the trip out to Austin to sit down. It's always more fun to do these in person.

Thanks for having me. We're going to take advantage of being in Austin.

Yeah, I know. This is a topic that we've received a lot of interest on, and people have always wanted to go deeper on this topic. My reluctance to do so has been, in part, driven by a couple of things. One is just my general lack of clarity around finding people that can speak about the topic with some degree of rigor. Then secondly, it's just a market of products that seem so sketchy. When you and I met, that's probably been six months ago and connected, we went for this walk and I was thinking, Oh, this is like the most I've learned about this and the rest is history. Here we are. Maybe give folks a bit of a sense of your background scientifically. I think when we met, we figured out pretty quickly we had both been at Hopkins at almost about the same time. But you, of course, were doing your PhD there. What did you do your PhD in again?

Yeah, my PhD was in biochemistry and molecular biology, so really thinking about enzymes and pathways and how they all interact with each other. Then I did a postdoc at Northwestern. We were looking for diagnostic markers for pediatric Wilms tumors. And then I moved out to Bayer and I worked in pharma. We were developing drugs for Parkinson's disease. I followed a pretty traditional path of a scientist into pharmaceutical drug development. Then I did what everybody does in Silicon Valley. I joined a startup company, and this was a DNA sequencing instrument company. And we went through rapid growth. We went public. And on the other side of that, I started this company, Pengylam. And it was really premised in the fact that things like probiotics and yogurt have been on the shelves for decades, but there actually hasn't been a new ingredient introduced in over 50 years. And that's because microbiome science wasn't a real science until DNA sequencing technology enabled us to really be able to survey the microbiome and create these metabolic maps and start to approach it like a biochemical problem and a systems biology problem. And all of a sudden it seemed like, wow, there had been a lot of unlocks in DNA sequencing technologies that would allow this entirely nascent field of science medicine called the microbiome to produce real products.

Here we are 10 years later in it.

We'll definitely spend some time talking about products and the evolution that you've had in that path. But let's talk more broadly, just about this field. First of all, how do we define the microbiome?

The microbiome is essentially all of the microbes. The bacteria, the viruses, fungi, yeast that reside in and on us. They are on our skin, they're in our nasal passages, they're in our lungs, and they're in our guts. Got it.

Now, our guts, which run mouth to anus, are outside of our body. People don't think of it always that way, but of course they are. What allows the colonization of that? I mean, is that something that is set at birth? Maybe taking a step back, when a child is in his mother's or her mother's womb, there's amniotic fluid that's flowing through that spot. Is that a sterile area?

Well, interestingly, for a long time, as you know, we've all believed that was an entirely sterile environment, and there were no microbes there at all. And some recent studies have started to elucidate that there are some strains, but it's very minimal. When we think about the gut microbiome of an adult versus somebody who's in the womb, it's incredibly much more diverse once you become an adult. And in fact, really, the primary initial seeding of the microbiome is through the delivery in the vaginal canal. And so we're going to get gross for a second. But literally, as you're being delivered, you are consuming fecal matter that is in the vaginal canal, and that's your first seeding of microbes. And infants have a very small diversity of microbes that are really tied to mother's breast milk. And then as you start to eat foods and as you start to get exposure to other environments, then the diversity for microbiome starts to really grow and flourish. And then at some point on the aging process, the opposite starts happening. You start to become less diverse in your microbiome as we age. So you start out almost like a blank slate, you get a lot more diverse.

And then as we age, you start to lose that diversity and therefore some key functions in the microbiome.

When is peak diversity? Approximately what decade of life?

Obviously, it varies from person to person. But if you can remember a time where you could eat or drink whatever you wanted to and you didn't have to worry about it, that would probably be the time.

Oh, so that's actually.

Quite young. Teenagers.

As a teenager, exactly. Yeah. When I could indeed eat a bowl of cereal for every meal with no consequence. By bowl, I mean a bowl the size of my head. So it's a box per bowl per meal. Okay, so we have a relatively early peak in life for diversity. You hear all of these bumper-stickers, slogans about the gut biome. Oh, it outnumbers us 10:1. Is that true?

I think those numbers have definitely come into question. They're nice to give people a framework for the fact that you have a ton of microbes in you. I think that's the important part, is that whether they outnumber you 10:1 or 2:1, I think is relatively probably not that important. But what is important is that they make up a huge portion of your body, mass as well as functions. And so it's an important key part of.

Your health. Call it even.

One to one. Yeah, even one to one is huge.

The idea is there are many cells that are not you between your mouth and your anus as there are you. Exactly. Now, obviously, just to have someone wrap their head around that, we're made up 70 % of water. So most of our mass is water, not the cells minus the water. Are these largely anhydra cells? How do they weigh so little relative to the rest of us?

Oh, man, I don't know the water content of bacteria, but maybe I think about it a little bit differently, which is more about the biochemical, so everything's going to come back to that. But it's more about the biochemical functions. What's the output of each of these cells versus the output of our cells? And I think when you look at it that way, these are real workhorses. So there's definitely redundancy among bacterial cells, but each of them is having multiple functions and multiple outputs. And so when you think about it at cellular level, I would think more about what are the things being produced by the cell. And bacteria tend to secrete a lot of things that they're producing, unlike the cells in our body. There's a lot of function that's associated with the microbiome that's super important.

I remember in my first and only biology course as a kid, because I didn't take bio again until I decided to go to med school in ninth grade or whatever. We learned about pro-cariots and eukaryots. I still remember to this day the joke of our teacher, Mr. Jefferson. He said, Do you know what a eukaryot is? Everyone was like, No. He goes, It's a portaging term. Portaging, when you carry canoes back and forth between rivers anyway. No, it's a horrible joke, but I still remember it. I'm going to have to cut this part out of the podcast. It's so bad. Where are we going? Where I'm going with this is, can you explain to people listening what the difference is between our cells and bacterial cells? Because there are some fundamental differences between these things called prokaryots and eukaryots.

Yeah. I mean, I think that, first of all, every cell in our body needs the other cells and the organs and the whole system in order to be able to survive and do their job. Whereas bacteria, they don't need anybody else. And so every bacterial cell, every unit is its own living thing that can replicate, perform functions, lose functions, be genetically modified, and all of that. And so it's its own entity, its own living organism. Every cell is a living organism. And then they divide really, really rapidly, some of them as quickly as 10-15 minutes you're dividing. And so there's this other component, which is that some of these bacterial strains, because they divide so quickly and because they're also under the pressures of evolutionary processes, is that they can evolve super quickly. And so we as humans, we have a long evolutionary timeline because it comes in the form of you make a kid who makes a kid who makes a kid. Now imagine if that was happening every 10 minutes, you can evolve really, really rapidly. That's part of the antibiotic crisis out there, which is to say that these things can become resistant to antibiotics because of this division time.

You alluded to this already, but they have the capacity to secrete things significantly. We think of bacteria and we hear it as a bad term. We think of a bacteria is a bad thing, and there clearly are some bad bacteria. But would I be oversimplifying if I said that most bacteria enjoy a complementary relationship with us as their host? Is that fair in terms of flora, such as the bacteria on our skin, in our nasal passages, in our gut?

We've co-evolved with these microbes and these bacteria. And so generally speaking, when you're co-evolving with something, there's some mutual benefit. I cringe when people talk about good bacteria and bad bacteria, although I do it as well, because it's the ecosystem and the context of these bacteria that's actually more relevant. A good bacteria can become a bad bacteria in a certain situation, and likewise, bad bacteria can become beneficial in a different context. And so I think that it's important to know that they're all part of these different pathways and what they're doing together. So, for example, Clostridium diphycyl is something that I think people think is a terrible pathogen, and it's so bad for you. Oh, my gosh, you better never get it. Almost all of us have Clostridium diphycyl in our guts. But at the level that it's at and in the context of the ecosystem of our strains, it's not having that really nasty pathogenic impact. In my opinion, there aren't really bad bugs.

People hear me rail about good cholesterol and bad cholesterol being meaningless terms. And of course, cholesterol is simply cholesterol. It's where it ends up that can be good or bad. So that's actually a great analogy. While we're on the topic of Clusteredium Difficile, which we'll come back to in more detail. What is the prevalence of that as a function of total gut biome in a person who's healthy and not otherwise in a pathologic state?

Well, this is the convoluting part about the microbiome, which is that if you sequence and do really deep sequencing and even biochemical assays across a person's microbiome, and then you say, right now I want to do population studies, what you find is that the difference between people is huge. And so the human microbiome project, in which they looked across 10,000 plus people at all ages and different demographics, really demonstrated that at the strain level, people are pretty different from person to person. When you start to look at the functions, that's where you start to see some redundancy. It's hard to say for a particular strain, if someone gives you an actual number and they don't give you a range, that's probably not correct.

Wow, interesting. Tell me more about that project. What were the observations it landed at vis-a-vis how various factors, both modifiable and unmodifiable, either genetic or age and diet being the most obvious modifiable factor, how did that impact the gut biome in these 10,000 people? Those differences magnify in the outcomes.

That initiative didn't have a longitudinal component to it or a perturbation of the system and looking at before and after. It really was just a- A.

One-time.

Observation. -one-time observation saying, Okay, we just look across a population of people. This is super early on, so we don't know anything about the microbiome. We barely knew how to sequence the thing. And even things like, well, what should the sample be? Should it be a scoop of somebody's stool? Should it be an entirety of the stool? Should you do 16S sequencing, which is just a gene that all microbes have? Or should you do the whole genome sequencing? That's going to be a lot more expensive. This is a government-funded project. And so there was a lot of unknowns at that time. So even just getting this information of if I looked across 10,000 people from skin to gut to vaginal microbiome, what does it look like? That was a huge endeavor. Of course, coming out of that have been a ton of longitudinal studies and studies where people have done actual interventions.

Going back to what you said earlier, it's not just bacteria, it's viruses, yeast, fungi. What does the pie chart of that look like in terms of numbers? Then what does the pie chart like that look like in terms of function? I'm guessing the bacteria are doing the majority of the work. I do feel pretty comfortable saying viruses are bad. Is there an exception to that rule? I mean, we certainly can harness viruses to do good things for us in terms of recombinant DNA technology. But if there was not a single virus on this planet, afflicting humans, I think we'd be in a better place, right? I'm not aware of good viruses.

Well, this field is really nascent. I hesitate to even answer the question of what is the role or the significance of all these different types of microbes. Interestingly, I think we also don't really know what the role of viruses are in the microbiome, but one could imagine that you have functions that are important and that you need to be healthy and that maybe these viruses help accelerate movement of those particular genes from one bacteria to another. I wouldn't say the jury is totally out of my mind on that.

Interesting. Yeah, that's a good point. Do we know anything about how our microbiome compares in complexity to that of other animals?

A lot of microbiome studies are done in mouse models. Having been in pharma, I feel like if you're a mouse and you've got cancer, you're made. We have so many different cures for you. The translation of that into humans is not that great. And the microbiome is even worse because animals eat different foods. Your diet is one of the biggest things that impacts your microbiome. And so now you're saying, what is an animal's microbiome? How does it interact with the host? And then what implications that I have for health? Or even one more step or move. There are a lot of strains that exist from mouse to man. But I think, again, because it's context, it's not totally clear that you can really use these animal models to predict what will happen in a human.

Has anybody looked at animals that are not living in captivity and not genetically ridiculous, like the typical black-sixed mouse and things like that? I mean, do we know what our pet dogs look like or what animals in the wild look like? Do they more closely resemble us? Or again, is it purely a function of what they're eating?

A function of what they're eating is such a big driving factor. If you look at your pet dog, I mean, it really does depend on what you're feeding them. Some people feed their dogs are pretty much a grain based diet. Some people are cooking chicken for their dogs every night. So when they look at pets, I think that's also hard because it just ends up being parsed out by what you're feeding them. Just to get back to the reality of these genetically modified mouse models, it's even more extreme in microbiome studies. They literally make, I call them like the bubble boy of mice. They try to entirely deplete these mice of microbiomes. They're taking a mouse that has no microbiome, and then they're infusing them with a human microbiome, and they're saying, Okay, what happens now to the mouse? And so these are germ-free mouse models that are really commonly used to understand the impact of a human microbiome on the health of the animal. This is completely not what would ever happen in real life.

For folks listening again who might not think much about bacteria, my vague recollection of microbiology is one line in the sand we draw to divide them is, are they aerobic? Are they anaerobic? Are they gram positive? Are they gram negative? Which is simply a staining technique. I guess I should explain that aerobic are bacteria that require oxygen for respiration. Anaerobic are animals that generate ATP without oxygen. Then you have facultative of each where they prefer to do it one way, but they can do it the other way. Any other divisions worth talking about as we explain the types of bacteria?

The aerobic versus anaerobic is definitely one of the most important things you think about. You've got bacteria on your skin that definitely loves oxygen. Then what we call the gut microbiome is actually a strictly anaerobic area of the distal colon. So there's literally no oxygen. Those strains can't even grow in the presence of oxygen. One other thing might be localization. So your GI tract and the gut microbiome, there's this so-called gut lining. So the cells, the epithelial cells where there's this mucin layer that's an important part of having a well-fortified gut lining, there are strains that live in that mucin layer. And so that's a different type of a strain. They feed off of mucin versus many of the other things that feed off of things like the foods that you eat or prebiotics.

How does the characteristic, let's just focus on the bacteria, how does the characteristic of the bacteria change from mouth to anus? Obviously, you're going into less and less oxygen as you go down. Presumably, you don't have pure anaerobes in the mouth. But I know from the little bit I remember about working in an ER, whenever somebody received a bite, you would think, How bad can a bite be? We were really conditioned to remember that those are some of the dirtiest wounds a human can have. No less dirty than a feces soiled wound. So even at the approximate end of that bacterial lining, these are really frightening bacteria. Can you tell me anything about how the bacteria change as you progress along the length of that? By the way, that's a very long tube. It's not just measuring here to here. The listener would have to understand how tortuous the small intestine and the colon can be.

The primary thing that changes is this anaerobic part. So obviously your mouth, there's a lot of oxygen exposure. And then as we said, when you get to the distal colon, there's no oxygen there. So all along that path from the mouth to the stomach, I think, is where you have a reasonable amount of oxygen exposure. Once something gets through the stomach, there's this in between the strict anaerobic and the aerobic area. And a lot of the Lactobacillus and Bifidobacterium strains that are on the labels of probiotics out there today, they reside in that small intestine area. And then you get to the recesses of the gut microbiome, which is where all the action happens. So after your stomach has broken down foods and they make their way to the distal colon, that's where an incredible amount of metabolism is happening. And there there's no oxygen. But yeah, the mouth microbiome is really interesting opportunity because what's happening in the mouth, the two ends that you mentioned, mouth and anus, what's happening in the mouth and what's coming out the other side? Give you an indication of what's happening in the middle. I'll just tell you a funny story.

Early on when I started this company, I met a guy who was really interested in hyena mouth microbiome. The reason is because the hyena is one of these bizarre animals that can eat a carcass of an animal after it's been dead for extended periods of time, a week, two weeks. No other animal go near an animal that's been dead for that amount of time because of the bacterial overload. This is just going to kill you. And so it's so-called rotten meat that these hyenas are able to eat. And there's this big question of why are they able to do that? And it turns that if you look at the microbiome of a hyena, they make an incredible amount of antibiotics. And so he had this whole theory that if you could understand what antibiotics are being generated in that hyena's mouth microbiome, it might be a new source of antibiotics for us. And he was so extreme in this belief that the hyena had such a clean mouth that in the context of doing this job of trying to get microbiomes. He got bit by a hyena. He went to the emergency room and they're getting ready to give him antibiotics.

And he said, No, I don't want antibiotics. It's going to decimate my microbiome. Furthermore, I know that because I got bit by a hyena, I'm not going to get an infection. They have the cleanest mouths and need to sign all these waivers. Apparently, he never got an infection. But the mouth microbiome is a source of potentially bacteria, but maybe other sources of new science.

What is the classification? What's the org chart of these bacteria?

Now we're going to get back to seventh grade biology where you have the phylum, the family, the genus, the species, the strain. That's the organization of them.

I don't even remember that anymore. Say it again.

It's the phylum, the family, genus-species-strain. And so what DNA sequencing has enabled us to do is to really look at strains. And maybe one interesting thing is even within strain identity, so we give names to these strains based on their genomic makeup, even that part is really evolving in our understanding as well. So, for example, methylation is a hot topic when it comes to humans. Bacterial genomes are also methylated. It's typically used for silencing certain genes. But you have this question of, is it really just the genetic makeup or these post modifications also changing a strain from one thing to another? Should you define a strain by its genomic makeup, which is the traditional way we define things? Or should you define it based on what it's doing and its actions, and that becomes more complicated? I think we're going to see that definition maybe evolve as we learn more.

Do you mostly operate in a world where you're thinking of the strain then?

Yeah, we operate in a world where we're thinking about the strain and the function. For example, as we manufacture our strains, we do occasionally these whole genome audits because these strains do divide and replicate, most of them basically every two hours. You want to make sure that whatever genetic modifications are just happening naturally don't actually impact the function of the strain. So we literally do these audits. We'll do a whole genome sequencing of these batches. We'll do a full panel of biochemical assays to understand, are they still generating small molecules at the rate that we'd expect, growth curves. And so I think that's one of the challenges to actually being in this area is that these guys evolve really quickly.

Yeah. I mean, it's hard for me to wrap my head around that because even though I acknowledge what you're saying with respect to the speed of their multiplication or replication cycle, when I think of my former life when I was in a hospital, everybody knows what Mercer is. But does it mean that there's like a new Mercer every pick your favorite period of time where even something every month there's a new Mercer in the hospital thing that fortunately always still seems to be responsive to vancomycin. But at some point, it won't be like... I guess there's VRE, right? Anx-resistant enterococcus. It's hard for me to wrap my head around the speed with which these things are mutating. That within the span of a year of your life, does that mean that your gut biome is changing not just as a result of you changing, but because they're evolving? I still don't understand exactly who's optimizing for what? Let me reframe my question. What are they optimizing for in their rapid evolution?

They're optimizing for their environment. Again, if you take diet as the primary thing that can modify your microbiome, it's the primary thing that can modify your microbiome because that's their food. They're living in your gut. They're waiting for you to feed them.

The reason that MRSA and VRE are evolving is to escape the antibiotic- Yes.

-which is against our best interest. But what you're saying is, at least if I'm hearing you correctly, it could be that the evolution of our gut biome is in our best interest because, in theory, it's evolving to its environment, which is us.

Exactly. It's evolving to survive what we're feeding them and the environment that we're creating for them.

Interesting. Okay. So very, very fluid.

It is. You can go on a trip to another country and eat the food there for a week and come back and your microbiome looks totally different in just a short period of time. I mean, many of us have experienced that. You go travel, you get GI distress from foods because you're not used to having them, and you can whole hog change your microbiome. That's actually what really drew me to this field, because when you're talking about human genomics and human systems, the way in which you can impact them is limited because it's an already existing system that's relatively immutable without some real serious external force. You have to introduce a chemical small molecule to change a pathway that has five backups to it in your system. But the microbiome is incredibly mutable, and we're doing it all the time. So when you think about the ability to change it and to have real health implications, that's where it's at. That's why it's an exciting field.

Got it. Makes sense. Let's talk about how one measures these things both in the lab, so what you would do, but also maybe, for lack of a better word, over the counter, what I could do. If we were interested in understanding your genes, we could do them in a number of ways. We could do the gold standard, which was we could do a whole genome sequence. We could sequence every nucleotide of every single coding and noncoding gene in your body. Twenty years ago, that would have cost close to a billion dollars. Today, that's about a thousand dollars. Still not the most practical test in the world because it yields a whole bunch of information one doesn't know what to do with. There's the gold standard. Conversely, we could go on a fishing expedition, and you could say, Well, I'm really worried about my risk of cancer. We could do a commercial test that looks at a whole bunch of known polymorphism snips, and we could screen for a hundred of those things. It's a much more targeted look, and we could get that information. Walk me through the menu of options that you, as a scientist, would embark on to do this, and then what a consumer can do.

It's actually quite similar when it comes to understanding the microbiome. You can do shotgun sequencing, where you're getting the entire genomes of all the different microbes, and then you need to be able to assign which genome goes to which microbe, and there's a fair amount of redundancy. You use long-read paired with short-read sequencing to be able to get-.

Tell folks what that is so they understand. Yeah, because it's a bit complicated.

It's exactly how it sounds. Short-read DNA sequencing gives you... Let's take an example. You've got 1,000 bases of DNA that you want to sequence.

A bacteria has typically how many genes and how many base pairs?

Really is a huge range of sizes. If you're trying to measure a certain piece of DNA, you can use short-read sequencing technologies, which allow you to, with high accuracy, get these short pieces of your sequence. You get maybe 200 base pairs, and so you would have a few of these and then you paste them together based on the overlaps. The overlaps, yeah. And each piece is pretty accurate in terms of the sequence. Then you can do long read sequencing, which will allow you to get in one shot that entire thousand base pairs, but that tends to be a slightly lower fidelity read, and so you might have some errors in there. That's why the best way to do it is to do both. I would say the reason that stitching together is problematic is because bacteria have redundant genes. Some bacteria might have five copies of a gene versus another bacteria that has 10 copies of a gene, and turns out it matters. I'm not going to remember the name of this bacterial strain, but there is a strain that's been studied pretty well that metabolizes Dysoxin.

A drug that's used to treat arrhythmias. Yes.

One of the reasons why that drug fell from being first line is because the efficacy had such a wide, broad range across people, so it's hard to know what's the right amount prescribed to somebody.

Wait, does it treat arrhythmias.

Or heart failure?

It's heart failure.

Yeah. That's how long I've been away from the game.

What they found was that there's a microbe that can metabolize Dysoxone. People who needed higher doses to have efficacy had higher amounts of this strain. But then in another double-click of that, it wasn't just that strain, it was how many copies of this particular gene that had. If it had over five, it could metabolize Dysoxone. Less than five, it couldn't.

That means that that bacteria exist high in the GI tract, presumably, because if that were just something in your cecum or beyond, presumably it wouldn't have impacted. I mean, it has to be somewhat in proximity to the liver. It has such an impact on the bioavailability of that drug, right?

Isn't digoxin in orally? Yes. I actually don't remember the location of this bacteria, but I remember this part about the number of genes being important as to whether it could even metabolize a thing or not.

Because normally we think a lot about the different genes in the P450 system in the liver, which is heavily responsible for how most drugs are metabolized. That clearly explains a lot of the variability in human metabolism of drugs. I never knew about this. This is even one step beyond that, and obviously not dependent on our genome, but the genome of this host.

Yeah. These are mostly gut microbes. It really is, especially for these oral drugs, there is some path that it takes for interacting with these microbes, and they are able to metabolize these various drugs. Getting back to your sequencing question, the number of replicates matter. If you imagine you have these short snippets, it's a guessing game as to, well, is this an actual just replicate or was it only there one time? Was it the second one? Exactly. That's why you need- A long sequence. -becomes your template to put these short reads on. That costs several thousand dollars per sample to do.

Even today.

Even today, because you're really trying to get a comprehensive. Then just imagine that, and now it's every bacterial strain out there. Ps, there's a lot of redundancy and bacterial strains. For a large number of them. We don't even know, much like the human genome, we don't even know, are these real bacteria? What do they do? There's just still a lot of uncatalog genes and so that's our role endeavor. The second way is to really look at a qPCR-based, where you're really looking at a specific strain and trying to understand how much of that exists.

Tell folks why the PCR would work that way, what you have to use and how you have primary-pronged numbers and why that's analogous to the, let's look at your cancer genes approach.

Yeah. You might have a lot of bacterial strains in your ecosystem, but you don't know how much they are relative to each other. You can get a catalog of everything that's in there, but you don't really know how much of each one is in there from these sequencing methodologies. Although there are some really interesting tools that people are starting to develop to try to get at that. But the more accurate way to get at the quantitation or how much of this strain is actually in the microbiome is this quantitative PCR. You basically make primers that are specific to that strain, and then you're using PCR in a quantitative way to understand, well, how much of it is in there compared to, say, some other strain or compared to the entirety of all the different bacteria in there. And now you have an idea of, is this constituting 1 % of my microbiome, 10 % of my microbiome? And so you get this quantitative piece. If you're low in specific microbes or specific functions, this becomes the way that you would really look at that. But there's an added very important complexity that the microbiome has that the human genome doesn't have, and that's around this replication and ever-changing thing.

And so it's really not good enough, as we talked about with the human microbiome project. It's not good enough to get one snapshot in time. You really want to understand what is your baseline microbiome. And then if you ever change your diet, you travel, you go on antibiotics, there's all kinds of things that can change your microbiome in very acute ways, all of a sudden becomes a different microbiome. And so this longitudinal data really matters. And then additionally, because every microbe is replicating at a different rate, the constitution or the fraction in which one microbe might be in your gut today might be different tomorrow. And so you need longitudinal data that gets you the quantitative piece plus who are the players. And then the third part is because they're also mutating, you need to understand what do the functions change. So if you really want to understand the microbiome, we were spending $5,000 to $6,000 per sample to understand longitudinally what's happening, what are the different functions? How are these things changing? It's pretty hard to do.

What are the ideal functional assays? Because I think it's important that we don't lose sight of what actually matters. It's what clearly matters when we look at our cells. I think we're really understanding that today in humans that function matters more than genome. The protein is more important than the gene. What do we look at in the gut biome? Are you looking at secretory products? How are you determining the health of the function versus just the genome, which obviously must be correlated with it, but not exact, right?

We are super interested in carbohydrate metabolism. And so we look at the output of that as the short-chain fatty acid. So butyrate, propionate, acetate. And so you can take an individual strain, feed it a substrate, depending on where in that biochemical pathway it is. Its substrate might be slightly different. And then look at how much of those small molecules are being produced on a gas chromatographer. And so you're running a enzymatic reaction. If you think about the bacteria as the enzyme, you're giving a substrate and you're basically doing your old fashioned Mikaela's Mennon curves where you're giving it increasing amounts of substrate. And you're basically looking at the enzymeology of going from substrate to that short-chain fatty acid.

Let's use that as an example. You eat a piece of bread, starch, polysaccharide, digestion, of course, begins in the mouth. Amylase starts to break that down. It continues further in the stomach. By the time it gets to, God, the jejunum, I mean, it's mostly just glucose monomers, right?

I think that's still being figured out, actually. What is the state of affairs when things arrive? I mean, it's actually really hard to survey what's happening in the gut microbiome because we don't have good sampling methodologies.

That's super interesting. You're saying even if you dropped a tube down somebody's throat and you just sampled the slurry at the distal end of the duodenum or the proximal end of the dujunum, I mean, I've seen what it looks like we see when you operate on somebody, we can't tell what the composition of matter is there.

I'm not squarely in this, but I haven't seen anything pop out that was really compelling. The pushback all of those technologies get is the ease with which things can get contaminated. Essentially, you might be able to get a sample, but then in the process of pulling that back out, it becomes contaminated with the other things that are along the track.

It could be done interoperatively. Hopefully, someone is doing that right. If you're in there operating otherwise... Now the problem is you wouldn't be operating on somebody without a bowel prep. You've completely destroyed the system.

Also, you're not operating on somebody in an anaerobic chamber.

The second they get exposed to oxygen, it's different. Explain how carbohydrate metabolism produces by-products and what those by-products are. Because people who listen to this podcast, people like me, when I think of carbohydrate metabolism, I don't think of any of the things you just said. I think of glucose. I think of glucose-1-phosphate. I think of glucose-6-phostate. I think of pyruvate. Ithink of acetyl-CoA, I think of acetylcholine. I think of the creb cycle. I think of lactate as a byproduct of oxidative phosphorylation. You're thinking of things in a different level because you're obviously looking at a different host. Explain the metabolism on that side of the ledger.

In this case, when we talk about carbohydrates, we're really talking about these fibers. Our microbiome is uniquely positioned to metabolize fibers, a wide variety of which we actually can't even metabolize ourselves. Fiber is one of the primary prebiotics that feeds all of your strains. And so you're right. When we think about carbohydrate metabolism from the perspective of the microbiome, it's really thinking about these fibers. There are primary and secondary fermenters in the microbiome that can metabolize these fibers into certain short-chain fatty acids, which then become precursors for the ultimate short-chain fatty acid, which is butyrate. Butyrate is this incredibly important short-chain fatty acid that's been studied in a wide variety of conditions. And so your microbiome, one of the most important molecules it makes is butyrate. And so butyrate has a role in GI health. The colon cells are the only cells that use butyrate as their source of energy as opposed to glucose, which is what used by every other cell. And so when you don't have enough butyrate that's been associated with things like colon cancer and not having good colon health. But butyrate is also a small molecule that triggers G-protein-coupled receptors to then release GLP-1 from these L cells in the microbiome.

So it also plays a role in this gut metabolism axis. Butyrate becomes this really important small molecule that the gut is producing based on the foods that you're eating. And that's where we've really honed in.

So if we think about the difference between soluble and ins soluble fiber, are you referring mostly here to ins soluble fiber? Because you alluded to the fact that it's fiber we aren't able to digest and metabolize, that is the foodstock for these bacteria?

Yeah. That's really where we're focused in on, is these ins soluble fibers.

Ins soluble fiber, I guess, is most readily available in vegetables, correct?

Exactly. Vegetables and fruits. Yeah.

I was asked recently by a patient, I drink this green drink every morning, and they're like, Why do you drink it? Do you think it's a substitute for having vegetables? I said, I really don't. I said, I think it's a substitute for having vegetables with respect to the vitamins you get and probably even a lot of the polyphenols. But it's clearly not a substitute for the fiber. Just based on the practicality. You can look at the ingredient label. There isn't enough fiber in this to be a substitute. At the end of the day, there doesn't appear to be, if you buy the argument, which I think we're going to discuss, that fiber is essential for gut health, which by extension, means essential for human health, you have to be eating a ton of fiber, ins soluble fiber.

Yeah, you do. Although, I mean, there have been a myriad of studies showing that getting fiber from these external sources, these powders, and you have to get them in the volumes needed, they can be pretty impactful for your microbiome as well.

What are typical recommendations of fiber? How many grams per day? 18, 30, something like that?

I actually don't know what the current standard recommended dose is, but I think it's somewhere between 20 and 30 grams.

Yeah, it's hard to imagine getting that in a supplement.

I think actually your average American is consuming 1-2. We're way off base on how much fiber we're consuming. But just to get to your question, though, there's also a delivery component to this too. Taking the fiber where you mix it in a drink might be a lot less impactful or you might need a lot more of it, I should say, than if you were to take it in a enteric coated capsule that got through the stomach acid and then closer to the distal colon, it's a delivery question. So you might be able to get away with a lot less.

Oh, interesting. Why is that? I thought that given that it's ins soluble fiber, it would be impervious to the gut and the environment all along the way. Is that not the case?

The measurements of these things along the way has been elusive.

As my daughter would say, it's us. It's us.

We include inulin in our pill an incredibly small amount. It's in the order of 1-200 milligrams. So it's definitely not at the dietary fiber level. But the reason we included is because when we did our preclinical studies, we saw that if we didn't include it, the strains really didn't colonize as well. So the concept behind that is that by having the inulin right there next to the strains, as soon as that capsule dissolves, these things start to get hydrated and start to become alive in very close proximity as their food. That's why I think about that delivery and that proximity question as being something we haven't really solved for yet.

Interesting. Okay, so you eat a piece of celery, which is basically all ins soluble fiber. You mechanically break it into smaller pieces, but functionally, it's still cellulose that's making its way down. At what point in the gut? Does it have to wait till it's in the colon or does the metabolism and production of butyrate begin in the small intestine?

There's some bifidobacterium in the small intestine that can help start to break that down. But I think the general thought is that the majority of that metabolism is happening in the distal colon.

Okay. Which strains of bacteria are most responsible for the metabolism of ins soluble fiber and the production of butyrate as a byproduct?

It's a multistep pathway. There are Clostridial strains which can do this breakdown. There are bifidobacterium strains that can do this breakdown. Then there is the strain Acromansia, which was discovered in the early 2000s. It also plays a really important role in generating these short-chain fatty acids.

Are these strains, do they belong to the same species?

These are the species names.

Okay.

Strain name would have two parts to it. If I said Acromansia mucinophila, that would be the name of the strain. But I shortened it to Acromansia, which really does represent the species, the higher level.

Got it. So E. Coli is...

Strain name.

-is actually a strain name.

Yeah. Achera, whatever it would be the species.

Got it. Clostridium is the species. Clostridium diphtheal is the strain. Yeah. Okay, let's pivot for a moment and actually talk about C. Diff. Because that's one of the most compelling arguments for an intervention in treating human disease by manipulating the gut bacteria. So tell folks what Clostridium diphtheal is, is a bacteria. And you already alluded to this, but how does it go from being a benign/benevolent participant in our co-existence with the universe to one that could kill us?

Clostridium disil is a strain that exists, and many of us have it in low levels in our gut microbiome, and we walk around perfectly healthy and fine with it. When you take an antibiotic, that's essentially almost like a nuclear bomb to your microbiome. It kills everything in there. But in some cases, it doesn't kill everything. And so there are these strains of Clostridium Dificil that after you take an antibiotic, it's killed the different strains off, but it didn't kill your Clostridium difficil strain. And the problem with killing off all the other strains is now all the competition is gone. So you imagine you have this ecosystem of different microbes, and now you've just gotten rid of all of them. So now you have this strain that has no competition and it can start to propagate unchecked. And so it's when you start to have these really high levels of this strain, Clostridium and Dificil, in your microbiome, that's when it starts to make you really sick, and actually, ultimately it's fatal. And so the way in which we treat infections is through more antibiotics. And so when you have this Clostridium and Dificil infection, which is a result of having taken an antibiotic, almost ironically, the cure also is another antibiotic.

And really what you're trying to do is to get an antibiotic that can kill that strain and get it back to its low levels until your microbiome can reconstitute itself through the food that you eat. The success rate of those antibiotics varies from person to person, but overall, it's something like 70 % successful. And remember, fatality is on the other.

Side of this. I was just about to say, make sure people understand what non-success.

Means here. Yes. 70 % success is not great when the 30 % means that you're going to die on the other side. One of the concepts was really to go in the reverse direction and say, Okay, if the problem is that now there's no competition and this guy can propagate unchecked, what if we just load up the person's gut with all these different microbes, reestablish an ecosystem, and then that's the way to temper this thing down. And that's called a fecal microbiome transplant. It's exactly what it sounds like. You literally take feces from a person and transplant it into another person. That thing has like a 99 % success rate.

When was this first done? I feel like this was at least in the 90s, maybe earlier.

Well, it was definitely done as one offs before people were doing real studies with it. Actually, a ton of them were done in Australia. It's like one of the places where the most of these have been done. But it has an incredible success rate. It's gross.

Yeah. What was it like back in the day and how is it done today? I assume today it's done through capsules.

Well, I think people are still using the methodology of actually going up through the colon. But people have tried to create capsules and tried to be able to make it so that you can just consume these capsules with a drink and then it gets there. Those seem to be effective. They're not as effective as the enema version. Because probably in the process of getting that stool into a freeze dried format or into a pill format, you're losing probably some of the diversity that you just get when you do it the old fashioned way.

There are risks associated with this or not? Are they overblown? There's certainly been hoopla about this at the FDA at one point. What was that about?

Fundamentally, it doesn't feel like a therapeutic to give people shit. I think there's this instinct that, okay, this isn't safe, but there's a reality around it, which is that the source of that person is really important. How do you know that that's a healthy, so-called healthy stool donor? And so what if they've got some weird pathogen that now you put it into this person who's already in a depleted state?

Who's already compromised?

Yeah. Then furthermore, because everybody's microbiomes do have these different functions, because it's possible that you might cure them of the C-diff, okay, great. And you might not have given them a new pathogen, that's fine. But you might have changed their metabolism of foods in a way that's not beneficial to them. And so, for example, there are some of these case studies of someone getting a fecal microbiome transplant, and now all of a sudden they have obesity issues that they never had before. What really is at the heart of it is that we're very early in understanding microbiome science. And so I think the FDA, their job is to make sure that people are safe. And so they have to decide, is it more safe to give people this cure that has this incredibly high success rate when fatality is on the other side? Or is it more safe to say, hey, let's keep learning more about this before we actually introduce it into clinical care? And they actually tried to shut the whole thing down. They essentially said, FMTs are not approved by the FDA. They're not safe until we learn more about the science.

What year was that ish?

This was probably, I don't know, maybe 20 years ago. Essentially, these people who had these physicians, these patients, patients, families who had all experienced this benefit. Then people who were in the queue to get it done, essentially with pitchforks and hand went to DC and said, This is crazy. This thing has such a high cure rate. How could you possibly take it away as an option for people? They put their tail between their legs and reversed it and let people do them.

Let's talk about an example you just gave there, which I know is theoretical, but I'm sure there are many cases like it where you take a lean individual who's hospitalized for some reason, they're given an antibiotic. In the process, they develop C. D. Colitis. They get a fecal transplant and they recover. The fecal transplant came from somebody who was obese, and now they develop obesity. The hypothesis here has to be pretty clear that the gut bacteria of the obese individual is playing a causal role in their obesity. I want to tackle that topic. But first, why wouldn't the habits of the recipient immediately override the bacteria that they got on the receiving end, given that their habits are more in line with being lean? In other words, given the rapid evolution of these bacteria, why do they persist in their phenotype?

Well, first of all, these are case studies. These are not clinical trials. I think you're dealing with the uncontrollable nature of humans. But I would say one of the interesting things is that these people's habits did not go back to what they used to have. And one of the things we're learning about the microbiome is through this gut-brain connection, your microbiome can actually influence your food cravings. And we actually have some data supporting that as well. And so if you think about this new microbiome as maybe it's not metabolizing food as efficiently, it gives you a predilection towards obesity. But on top of that, it changes your food cravings. You've actually now got a double whammy against you that keeps you from being your old self. So I think the microbiome, this gut-brain connection, its ability to generate neurotransmitters, its ability to change cravings is still at early stages of understanding, but it can basically change your behavior. So you're not really the old person you are.

Profound. Two thoughts. The first is, could you be, in that situation, forced to eat your old way? You're going to go to a one week boot camp where you're literally force fed. Everything you use to eat, would that be sufficient to return your gut biome to where it was? Talk about how the gut and the brain connect. So my vague recollection is serotonin plays a role here. You've already talked about GLP-1. Gip typically is secreted higher in the gut, but that presumably plays a role as well.

Again, this is in the early stages, and we're going to learn so much more over time. But the gut is a massive producer of a bunch of things that we've traditionally thought of as neurotransmitters. So, yes, serotonin, dopamine, GABA, this GLP-1, GIP. And then I would also say there are neurons in the gut. And so there's this relationship between the neurons in the gut and the neurons in the brain. The neurotransmitters being generated by the gut can make their way straight to the brain through the vagus nerve. And so there is potentially an opportunity, for example, to tackle stress and anxiety through the gut microbiome, where you have these gut microbes that can produce large amounts of GABA, which has been shown to be able to reduce stress and anxiety. And so is there an opportunity to go after the gut to reduce stress and anxiety? And again, getting back to this craving thing, it's really interesting for anybody who's ever felt stress or anxiety. Actually, your food cravings oftentimes change and are hand in hand with that. So this part about the gut-brain connection that involves both cravings and these neurotransmitters, I think, is really, really fascinating.

How much of that do you think is regulated by things outside of food? Meaning the gut flora, as it comes to changing function, which then changes our physiologic state. I mean, we've talked about food, we should talk about it some more clearly a very important piece. You've already talked about antibiotics. What are other modifiable factors that change this?

Certainly, the two biggest changes in your microbiome are the things we've talked about, antibiotics and nutrition. But beyond that, what we know is that there are, and this is especially with regards to diversity and loss of function of your microbiome. We know that as you age, your microbiome starts to become depleted. We know that when you go through periods of intense stress, your microbiome becomes depleted. We know that when your circadian rhythm gets disrupted, so you travel and day becomes night, night becomes day, that changes your microbiome.

By the way, when you say depleted, tell me what that means in a technical sense.

It means that what happens is that you have loss of function. So you start to become depleted in specific strains that have specific functions. And so, for example, this strain that I alluded to earlier, acromansia is one of the ones that is deeply studied. And low levels of acromansia have been associated with a really wide variety of diseases, ranging from obesity to immunological disorders to stress and anxiety and GI issues. Basically, the depletion means you're losing certain strains and certain functions. Then for us women, when we go through menopause, there's like a massive change in the microbiome on the other side of that lovely experience.

I was going to ask you about the vaginal microbiome, but right now, are you saying that with respect to gut as well?

I'm saying that with respect to gut. Yep.

Okay. What are the factors that influence the vaginal microbiome besides the most obvious, which must be menopause, pregnancy, or anything that has a dramatic shift in endometrial lining and changes. Presumably, if you sample a woman's vaginal microbiome across the month of her cycle, given that her estrogen, progesterone levels are fluctuating, you're going to see differences. But I assume there's a healthy versus an unhealthy. How does that manifest itself? And what does one do about that?

Well, I'm not an expert in the vaginal microbiome, but certainly these Lactobacillus strains are some of the more important strains in the vaginal microbiome. The name of the game there is actually production of acid. You want an acidic.

Environment there. It's a protection environment. It's a protection environment. The goal is to keep yeast, in particular, and.

Other things away. Exactly. Sperm, everything. There is a healthy vaginal microbiome, which is really associated with the acidity level that these bacteria keep the vaginal microbiome at. It can be influenced, actually, also by the food that you eat. I don't know. It could be that I don't know, but it's not totally hallucinated. But for example, you might know that one of the things that gets recommended for you, day is cranberry juice, and you're not inserting that into your vaginal canal, you're consuming it. And so there's definitely this connection between the food that you're eating and then the composition of your vaginal microbiome pregnancy is also another thing that really changes it. And one of the implications that we did look into, this is some work that we were doing with the Mayo Clinic early on in our company, is the idea of preterm labor. So preterm labor has been highly associated with bacterial vaginosis. And that is essentially a broad name because I think we don't fully understand what it means, but it's an infection or disruption of the vaginal microbiome. This infection triggers preterm labor. One of the ideas here is that, well, if you could prevent bacterial vaginosis, you might be able to reduce the incidence of preterm labor.

There's other idea, which is that once you have a preemy, you're a lot more prone to have a preemy again. These women usually also have this bacterial vaginosis, which happens right before they deliver the baby. Maybe there's something about your vaginal microbiome that makes you more susceptible to bacterial vaginosis, which then leads to this preterm labor. Then there's, of course, the UTIs and yeast infections and things like that.

Any other systemic complications from a negatively altered vaginal microbiome? I guess you could argue preterm labor is systemic. Outside of the pregnancy case, if you just talk about a woman who's entered menopause, because I'm guessing being on hormones versus not on hormones is going to have a profound impact on that. Do we know if any of the problems associated with menopause beyond the obvious ones like vaginal dryness and hot flashes, have their roots in a change in that microbiome?

I think that work is still pretty early on, so I would hesitate to go too far there. And even the gut microbiome, too, there are microbes that can... I'm not an expert in menopause or these hormones, but I believe that estrogen has a modification that gets made that is a signal to the body to not put that estrogen back in circulation, but to, I guess, you must urinate it out. But there are microbes in the gut, their job is to remove that modification. And so the effect is that it could potentially increase your circulating estrogen. And so this is all super early stage. If you go try to look up studies around menopause and the microbiome, it's extremely sparse. I think we just really don't know.

But there could be a day, presumably, when there are bacterial products that are used to address some of these issues. It certainly would make sense. Going back now to the example you gave, how robust is the evidence in humans? Maybe we can start by the evidence in mice because it gets all the headlines, where you can take an obese mouse and you can do a fecal transplant from a lean mouse into an obese mouse and make that obese mouse a lean mouse. Is that a relatively robust reproducible finding?

Yeah. A lot of people have done that. Even to take it one step further, they can make these mice obese or lean using a human microbiome transplant. They basically would take the microbiome from an obese woman, put into a lean mouse and see that lean mouse become obese. We talked about the overlap between mice and men. There is some overlap there because you can actually change the metabolism of these mice through these fecal transplants.

Why has that not taken off in humans? What are the impediments to that? Are those studies being done?

People have done fecal microbiome transplants into people with type two diabetes, for example. I think there's two big challenges. One is, of course, the safety challenge, which is, do you really feel confident that that stool donor is the stool that you want to have in your body and move forward with. But more profoundly and maybe linked to that is the ability to reproduce the effect. So even if it's always Joey who's providing the stool, I mean, Joey travels, Joey goes on antibiotics, Joey changes his diet, you may not always be getting the same stool from that donor. And so in order to generate consistent results, we do in drug development, dose-response curves, you're really trying to understand in this small molecule, in this how much of it do you need and what effect will it have, you can't do that if your intervention is a fecal microbiome transplant because that thing is different from person to person and even from the same person from time to time. It's a pretty messy drug. It's a messy drug. When we started this company, the idea was the fecal microbiome transplant is super compelling data. That tells you that there's something in that microbiome that can change your physiology.

But the name of the game is, how do you figure out what are the components of that microbiome that are conferring that benefit? And how do you make a reproducible, manufacturing, and quality process so that you know, every time I deliver this pill to you, I know exactly what's in it, and it's the same every single time. And so that becomes a challenge. How do you take that whole kitchen sink, the whole fecal microbiome, and say, actually, it's these four strains or these two strains or these 50 strains. That's the tricky part.

Let's talk about what those strains might be and what their function is. For folks listening, what are the strain names that we have to know here? Lactobacillus, you've already talked about. Acromancy, you've already talked about.

Yeah, I think if you start to look at labels on the supplements that are out there, you're going to see a lot of Lactobacillus and Bifidobacterium. Those are the two primary species that are pretty much in every product. Those strains are relatively easy to grow. It's one of the reasons why those are the strains that are in a lot.

Of probiotics. Why are they easy to grow? I thought anaerobes are hard to grow.

These fall into that in between their facultative. You can grow them in some amount of oxygen. In fact, an acromancy is not. It's a strict anaerobic. In fact, when we first started wanting to manufacture Acromancy, we just tried to outsource it to all these probiotics manufacturers around the world, and they kept sending us back dead stuff. It's because in order to manufacture a strict anaerobic, your entire manufacturing system end-to-end has to keep all oxygen out. A single molecule of oxygen kills the whole batch. There's a real manufacturing challenge to some of these next-generation probiotic strains.

Before we talk about that, let's talk more about the bifto and lacto. Are they a bit of the shiny light problem? The key is under the shiny light? Yeah. Yeah. Are they basically the ones that are most ubiquitous because they're easy to grow, or is there a really good physiologic case for their utility?

They're easy to grow and so they're prevalent. That's why so much work has been done on them. Because there was no microbiome science when these strains started making their way into the market in the '70s. There was just, what can I culture? And that's what's culturable. Nobody thought about strict anaerobes or how to keep oxygen out. That's a pain and that's expensive. So these are things that have been around since the '70s. And, furthermore, they got grandfathered in by the FDA as safe because they've been on the market for so long. You want to bring a new strain to market. You have to go through the entire process of the FDA that says, Hey, it's a new strain, but let me demonstrate to you all the thousands of reasons why this thing is safe.

And just to be clear, from a regulatory standpoint, if a company wants to put a new strain into a probiotic, they're going down a grass pathway, a generally regarded a safe pathway, or an IND?

If you want to be able to sell it directly to consumers- It has to be grass. -you have to go down the pathway.

Are there people that are actually looking at a pure pharma strategy where they're making an IND and going down the drug pathway?

Absolutely. There are pharmaceutical... Actually, they're all startups, but pharmaceutical drug companies that are using the microbiome as the therapeutic and filing INDs and doing these studies. In fact, this fecal microbiome transplant in the form of a pill, that's a therapeutic. There are multiple companies out there that are developing that as a drug therapy.

Interesting. I mean, the closest thing that I can think of to that would be immunotherapy using TIL. You have immunotherapy in the form of a drug where you use something like K-truda, C-TLA-4 inhibitor, where you have a single molecule that is an actual drug that triggers the immune system to do something. But then you have tumor infiltrating lymphocytes where you go and harvest a patient's tumor, grow those T cells out, multiply them a log or more, and then re-infuse them, actually several and then reinfuse them. But of course, then every patient has their own design or drug. That's still a purely regulated FDA, IND problem. That sounds a lot more like the fecal microbiome transplant, which is every patient has a different drug. The difference there is it's not coming from them. By the way, begs the question, should every patient going to the hospital have a banked stool sample so that they can be their own transplant? You could have an autologous transplant and save a lot of the risk.

Yeah, I think that would probably be a super smart thing to do. Bank your own stool. You could do it in your own freezer.

Right.

Just.

Leave a stool sample in your fridge when you go to the hospital. And if you can make it home and you're fine, you can pitch it. Yeah. Yeah, interesting. What drew your interest in this other species called acromansia?

I think that acromansia has become an incredibly interesting strain over the last decade. But when we first became interested in it, it was really centered around trying to understand the difference in people with metabolic syndrome versus people who don't have metabolic syndrome. We did, and a variety of people have done these twin studies. These twin studies are really interesting because you take a twin pair and you're looking for discordant twins where one twin is healthy, the other twin has diabetes or is obese. And you're looking at their microbiome and you're basically saying, Hey, man, genetically, these two people are the same. This is a nurture, not nature problem. And so you start to look at their microbiomes and you start to see patterns around the world. If you compile this data together, you start to see that the twin that has obesity or type 2 diabetes has a different microbiome from the twin that's healthy. And one of the hallmarks that appears to be true across different cultures and different dietary patterns is this depletion in this strain called acromansia.

In absolute numbers.

In absolute numbers.

And what's the relative difference between these populations?

That I don't know, but I do think that it is interesting because across these different populations, acromansia is, we don't have to get into this, but the so-called healthy gut microbiome, which is yet to be defined across the world, acromansia comprises the microbiome on the order of somewhere between 5-10 % or 5-8 % for a healthy individual no matter where you're living. And so being depleted or having nothing show up is a really stark difference.

Oh, it's that profound a difference. The unhealthy person has none of it. The healthy person is 5-10 %.

Yes.

At 5-10 %, does that make acromansia the most predominant species in the gut?

I think it is among the most... I actually don't know that there's a current telling of winners because there's such a difference from person to person in terms of the composition of the microbiome.

Now, you said something a second ago that's very interesting, which is you said, Regardless of where a person lived, if they're a very metabolically healthy individual, I'm paraphrasing a little bit, they're about 5-10 % acromansia. But presumably that also means it's independent of diet because you can point to people who are on radically different diets who are metabolically healthy. So how is that explained? You can have people who are on an all-plant diet who are metabolically healthy and presumably eating 100 grams or more of fiber a day, plenty of food. And you can have somebody on a carnivore diet. And I've seen people on these who are very metabolically healthy, I think it's impossible diet to adhere to for someone like me who are not eating a gram of fiber, but by all measurements are metabolically healthy, and then, of course, everything in between. So given the sensitivity of this flora to diet, how do we make that explanation?

I'm trying to think about any studies that really got at the heart of this. Really, the study that you would want to do is you want to take one of these individuals, measure their microbiome, and you'd want to put them on a series of different diets and just see like, is this something that is not changeable? No matter what you eat or what diet you're on, this particular strain stays high.

In which case you would argue that the microbiome is a readout of their metabolic health, not the cause of their metabolic health.

Could be. I was going to go in a slightly different direction, which is to say that perhaps the host themselves play a much larger role in the composition of your microbiome than we really understand. As you change your diet, you would expect your microbiome to change. But if there's something about the host, that part never changes, that might be the influence. To be honest, I just don't think we know.

Wow. I mean, that's interesting. By the way, it's possible that these studies haven't been done across a broad enough dietary spectrum. So even though there's geographic diversity, maybe there isn't as much dietary diversity. That would be interesting as well, I suppose. For example, not that we could go back and do this, but if you go and look at the Inuit who prior to the adoption of Western food, were eating a seemingly ridiculous diet, and yet were quite metabolically healthy.

Yeah. It does make me wonder if I go back and look at this too. I mean, there are tribes in South America that have been relatively untainted by anything outside of where they are. And so I know that there are definitely microbiome studies that are happening in some of those tribal groups. That could be the beginnings of this study.

Quickly, the difference between a probiotic and a prebiotic.

Oh, boy. Yeah, the vocabulary lesson. I'll introduce another one that's become popularized, which is the postbiotic. The microbiome, when we talk about it, is all these bacterial and yeast strains. That's the probiotic. The probiotic is the living organism itself. Prebiotic is the food that feeds those organisms. We talked about fibers and inulin and polyphenols and things like that. Those are prebiotics. Prebiotics are the food, the probiotic is the organism itself. Then what these organisms produce or what they secrete is now being called the postbiotic. You would call would be a postbiotic. Butyrate would be a postbiotic. Yeah. Then maybe one more term people might start seeing is synbiotic. All synbiotic refers to is you've mixed two or more of these things together: the pre and the probiotic together or the probiotic and the postbiotic. A synbiotic has multiple.

When people are consuming yogurt, they're consuming a prebiotic, presumably. It's the bacteria in it.

That they're trying to- Yeah. There are Lactobacillus bacteria that are in that yogurt that stay alive in the context of yogurt. That's really what you're consuming.

What is the perceived, believed, or realized efficacy of consuming massive amounts of Lactobacillus and Bifidobacter doctor?

I think the most well-documented and even reported from consumers impact of consuming Lactobacillus and bifidobacter. Probotics in general and yogurt is around GI symptoms. Things like gas and bloating and diarrhea and constipation, a lot of people report and there have been studies showing both sides of it. But basically, there have definitely been studies showing and people reporting they have better GI when they're consuming these probiotics or these yogurt with probiotics.

If you go and buy yogurt off the shelf, how much lactobacillus is naturally within that?

I think they added in, so it varies.

It's not something that naturally occurs in yogurt.

I think it is naturally occurring in yogurt, but all the things that you're buying off the shelf, they're also supplementing with additional lactobacillus. The real question to ask yourself when you're buying yogurt is how much sugar is in it.

Yeah, for sure. If you're buying fruity, flavored yogurt. By the way, I want to come back and talk about sugars in a second. But I want to go back to this probiotic. How much? What is the dose effect? I know that if you look at a bottle of pick your favorite probiotic, it usually uses something called CFUs, colony forming units. Can you explain what those are?

Yes. Some brilliant marketer decided that that was going to become the metric, the name of the game for probiotics. So colony forming units, remember back to seventh grade biology where you might have been given a petri dish and you had to swab your mouth or swab your hand or put your finger on it and then you see what grows. That's basically that tool. So you take your pill or whatever your yogurt, and you basically spread it out on a petri dish, and then you count how many colonies form, and that gives you a number. So you'll say, well, gee, this pill has 10 to the ninth colony forming units in it. So some marketer decided that that's the most important thing is the number of colony forming units, and I have 10 times more colony forming units than somebody else. Maybe that's interesting, but it's less relevant than the function of what's happening in your pill. And moreover, it only gives you one piece of data about what's in that pill. So when you do that, the only thing you get to see are what's able to form a colony. But actually, in almost every supplement, every probiotic out there, the majority of what's in that pill is dead probiotic.

And you don't see any of that when you use this technology. There's a different tool that can be used called flow cytometry. Essentially what you do is you take your capsule, you put it into this flow cytometer. And what it pops out, it's readout is live cells, dead cells, and in-between cells. It's based on a staining of the membrane. And so it tells you which of these cells is viable, the membrane is really intact, which of them have a compromised membrane, and which ones are somewhere in between. Now you know exactly what's in your pill. Because even if you could have the same number of live cells, it turns out that those dead cells and those in-between cells, they actually have a role to play. They have a function in there. These so-called postbiotics, that's what those guys are. You don't really know what's in your pill unless you're using flow cytometry versus.

Colony from the. When you do flow cytometry, what do you stain for? Which surface receptors or molecules are you staining for?

I don't know the answer to that. I think there's two that we stain for, but I actually don't remember.

Okay. And it varies by.

Bacteria, I'm assuming. Yeah. Well, there's some of these common ones among almost all bacteria, but yeah.

Lacto, it's basically able to use oxygen and not use oxygen. So obviously if you're going to produce it, you're going to produce it under the conditions of oxygen because it's cheaper and easier. I hear things about some of these things need to be refrigerated, some don't. What's the specificity of that?

Yeah, it's really about stabilizing something that's meant to be inside the body. And so I think that a lot of these Lactobacillus and Bifidobacterium strains, because we've been manufacturing them for so long, we figured out how to get them to be stable through this process. So just to maybe take a step back and what is the process, you take these strains and you're basically growing them into these big vets. If you've ever been to a brewery or a vineyard, you see these big vets, the same in these manufacturing plants. You grow these strains in a culturable media. And then when they get to a certain density, optical density, you then harvest them. So certification is one of the most common ways. You basically spin down the cells, get rid of the media, and now you have this paste of cells.

How slowly do you need to spin them to prevent them from dying?

Lactobacillus and bifidos, at least the ones that are on the market today, they're pretty hardy. We can spin them awfully fast and be done with this whole process pretty quickly. Then there's other strains that are a lot more sensitive, so you have to do this process much more slowly. I think that depends on the strain. But you essentially throw away the media, you take this paste of cells and then you freeze dry them. That gets them into a powder form. Once they're in a powder form, they tend to be pretty stable. Then you could throw them in a pill and you're off to the races.

What's the yield on that? How much loss do you have in live bacteria? Assuming you start with 100 % live in the paste, when you just go through the act of freeze drying them, what's your yield?

That is where everybody dies. Basically, a lot of times you're losing 90 % of your cells just through that freeze-drying process. It's a pretty harsh process. What people do is you try to figure out what cryoprotectants you can add that are not going to harm the bacteria on the other side of it, but that they're going to help them through this cryo phase.

How does freeze drying actually work?

It's both pressure and temperature. There are these instruments called lyophilizers, and a lot of them use these flat pans. You would put your paste... It's like baking. You put your paste in this flat pan, you slide into the lyophilizer, and then over a period of time, it's basically using temperature and cold, the sublimation process of removing the liquid from the cells directly into a gas. Again, we're going back to seventh grade biology. On the other side of that, you this powder. That powder may be stable at room temperature. It may still need to be refrigerated. But what you're trying to figure out is what are the additives that you can incorporate that help to get through this freeze-drying process and remain viable. But the things that have to stay refrigerated, even after that process, they're still relatively unstable, so you still have to keep them in the refrigerator. For us, for example, when we first started making Akkermansia, it absolutely had to be refrigerated. Within hours, it would die.

Meaning once you freeze dry it, you shut its metabolism down completely. Then the minute it gets even a little bit warmer, it basically warms enough to the point where it's no longer cryogenically preserved, but there's no substrate for it, and it dies. Is that the actual mechanism by which it dies?

That's how it dies, exactly. Because there's no substrate for it, and so it can't handle the heat, and so it dies. The most important part is you do want it to be able to get through this freeze-drying process. And of course, everybody wants shelf-stable product. But the most important thing is that when you ingest this strain, it actually makes its way to the gut microbiome is able to reconstitute and to perform its functions, secrete the proteins it's supposed to secrete, secrete the small molecules it's supposed to secrete. And so when you do this process, every time you make a model, you're like, Oh, my gosh, we just improved the viability for X. You just have to go to the other side and say, okay, now I got to put in a human and see if I got the same output. So it's a pretty lengthy process.

By the way, do you think that that 90 % loss is typical on the most common strains that are used commercially?

No. I mean, we even grow some of those strains and you can maintain higher viability. You're never going to get 100 % through, but I think it's more like on the order of between 50 and 80 %. You're definitely above the halfway mark.

Then how many of those make it into the person? In other words, once they're preheated, how many of them die along the way?

A lot of it depends on the encapsulation that you're using. If you have these enteric coated capsules, you can make the stomach acid, and then you can use these time-release capsules so that it takes a certain amount of time and you're making some assumptions around GI transit. Then there's the least expensive version, which probably all falls apart in the stomach and very little of it gets to the.

Actual destination. It wouldn't make sense to be drinking a probiotic and a liquid then?

You could get a lot more efficacy if you took it in a pill format. Part of it, too, is we talked about these Lactobacillus that are in yogurt. Part of it is that if you just have to get just on the other side of the stomach and the small intestine, that is a bit different thing than trying to get all the way to the gut microbiome.

Is that where it seems that Lacto and bifido need to be seeded? Is just outside the stomach?

I don't know that the answer to that is known, but you can actually find them all along the tract and even into the distal colon.

What is their functional output? Do they make butyrate?

Lactobacillus tend to produce lactic acid.

Lactate, besides that, yeah.

Bifidobacterium, various of those strains can produce short-chain fatty acids. They need to be paired with secondary fermenters in order to get to the final butyrate.

Now, lactate is also a great substrate fuel for entrocytes, correct?

Yes. I think maybe that's why these exist all along the track.

In that sense, is the primary function of the Lactobacillus to make food for the gut?

I don't think we know exactly the answer to that, but definitely the products of those Lactobacillus strains, there are a bunch of other strains that are dependent on having Lactobacillus.

Is it also in that 5-10% prevalence?

I actually don't know the answer to that. Okay.

Lots of companies out there are selling Lactobacillus probiotics. You should put them in the fridge, and you should hope that their manufacturing process is such that you're getting 80% instead of 10% of what they claim.

Yeah. This is where I think our government could maybe have an important role, which is to put some guidelines and requirements around labeling. I'll give you an even more crazy thing. Even the CFU, let's say you think CFU is a perfectly fine metric, the CFU that gets put on that label could be the CFU at the time of manufacture, or it could be the CFU. If I say this thing's got a two-year shelf life stability, it could be the CFU at the two-year time mark. You're really depending on, is this company going to tell me the higher number, or are they going to try to legitimately tell me what the thing is at the end of the shelf life?

Is CFU, by definition, always alive? Because presumably, if they form a colony, they were alive.

Yeah.

Are there any recommendations one would make about how many CFU you need of each of those two bacteria?

Well, I think people tend to converge around this like, Oh, you need billions. I don't know. I guess maybe I would ask the question in a slightly different way, which is, what are you trying to do? What problem are you trying to solve? What problem are you trying to solve? Exactly. If you've got a problem you're trying to solve, I generally think most people should operate under, I just need the minimum viable product to solve my problem. For you, that might be 10 billion. For somebody else, it might be 10 to the seven. Unfortunately, I think because of marketing, a lot of people are taking things and they don't even really know why they're taking them.

Well, it gets difficult when you don't know what the problem is that you're trying to fix. I think that therein lies a big challenge, which is, what am I objectively fixing? Now, it could be something symptomatic, as you said, it could be, boy, if I take a probiotic, my GI symptoms vanish. I'm not bloated. Great. Just titrate to the point where that's true. Going back to antibiotics for a second. For most people, taking antibiotics is almost always oral. But of course, if you're in the hospital, it's not uncommon to take an intravenous antibiotic. For example, if you're having surgery, it's not uncommon right before the surgeon cuts your skin for them to administer an intravenous antibiotic, usually something that covers for skin flora and that reduces the risk of surgical wound infection. Do intravenous antibiotics also have the same obliterating effect on the gut biome that oral ones do?

I have not investigated that, but these Clostridium diphyseal infections arise a lot of times when people are getting antibiotics that they're taking prior to surgeries.

I'm sure now that I think about it, people do get C. Difcolytis from IV antibiotics. That's a good point. What do we know about the ideal response to helping your gut out when you take antibiotics? This past year, I had to do two courses of Augmentin, which is a pretty powerful antibiotic. Kicking and screaming, I did it, but I had a pharyngeal abscess, and that's a no messing around type of infection. I'm on steroids and Augmentin trying to avoid surgery. Luckily, I did. But truthfully, I did nothing after the fact. I seem to feel okay. Am I just lucky or should I have done something?

We know that you have a ecosystem in your microbiome. You take an antibiotic that pretty much kills everybody. And then over some amount of time, you get a new microbiome from the food that you're eating, the environment you're in. And for a lot of people, that new microbiome is the same as your old microbiome. But for a lot of people, it's not. And one of the really interesting things are studies that have been done in kids where they show that kids who are systematically on antibiotics later on in life are more prone to things like obesity, type 2 diabetes, even things like ADHD, celiac disease. And so there's something I think, in those developing years where post antibiotic, you've got a different microbiome than pre-antibiotic. So that's just something to consider on the other side of an antibiotic. If ever you were going to clean up your diet, that's a good time to do it because you're starting with a new blank slate of your microbiome. And I think what happens for a lot of people is post antibiotic, they're actually starting to feel better and feel well, and then they're craving foods that maybe they haven't been able to eat.

And so you maybe aren't giving yourself the best shot at a good microbiome on the other side. I used to believe that taking a probiotic during an antibiotic course seemed illogical because the antibiotic is just going to kill that probiotic. But there have been studies coming out showing that, I think in particular, this study that's really compelling showed that if you did a fecal microbiome transplant after taking the antibiotics or at the tail end of that, or if you were taking probiotics during the time of taking antibiotics, that somehow on the other side of that, you had what we'll call a healthy gut microbiome. So you're to have some of these functions that we've talked about, the development of short-chain fatty acids. You've got Akkermansia in there. And so the idea here is that even though that antibiotic is killing these strains, you might be doing some a seeding at maybe very low undetectable levels that's allowing you to have a healthier microbiome on the other side of the antibiotics. So I guess if I were going to go on antibiotics, I would probably double down on the probiotics that I'm taking. But again, I think on the other side of that, really coming in hard and strong with a high fiber diet would be your best chance of reconstituting with really good microbes?

Maybe a naive question, which shows how little I understand microbiology. Antibiotics seem very specific. When you take Augmentin, you're really targeting a certain set of antibiotics. Oh, this really targets this gram-negative strain, and this really targets these gram-positive bacteria. That's why there are so many different types of antibiotics, right? I mean, it's not just the class of drugs like Cephalosporins. You have the first gen, the second gen, the third gen. At one point, I actually remembered what they all did, which I don't anymore. But the point is there was remarkable specificity. So why is it that if you just take one antibiotic for your skin infection, which presumably would be like a first-generation Cephalosporin, it obliterates your gut microbiome, which by the way, has nothing in common with the bacteria on your skin. But we just described that these are all highly anaerobic bacteria versus these guys that are totally aerobic. Why does an antibiotic even mess with the gut biome?

Yeah. I think antibiotics are a lot more broad spectrum than maybe people or the physicians that are recommending them know. I mean, yes, there's the gram negative and gram positive, but really the name of the game for people who are manufacturing and producing antibiotics is it's actually more useful to create a broad spectrum antibiotic because you want your antibiotic to be the one, the go-to-of-choice from a doctor. And if it is one that can tackle most different kinds of infection, you're going to become the more popular antibiotics. This is actually one of the arguments in the antibiotic world, which is that antibiotics over time have become more and more broad spectrum. They kill more and more different things, whereas you really want to go in this opposite direction, which.

Is- You want precision antibiotics.

-have a lot more precision around them. There's all these phage therapies and things where people are trying to get early specific, but that's new. Most of the antibiotics out there, they decimate your microbiome. They don't kill everything, which is why you can have these things like these C-diff infections.

Do you have a sense of just quantitatively? You took a gram of stool from me now and looked at how many bacteria were in there, and then you took a gram of stool from me after I was on an antibiotic for 10 days like augment and very broad, powerful antibiotic. What's the log fold reduction in viable bacteria?

I actually don't know the answer to that. That's probably published in lots of places.

Yeah, interesting. Okay, so what I'm hearing you say is the best thing you can do if you're on an antibiotic is, take advantage of the fact that you're starting with a new team and go out of your way to eat the best possible diet. We're not really talking much about specifics outside of fiber, are we? What I would do now, knowing this, what you've said is, I already love eating salad and vegetables and fruits, so I'd probably go on the all salad, all fruit diet for, I don't know, a few days. I mean, that might sound dumb, but I would go overboard on those things. Are there other things you would be mindful of, either eating or not eating during that recolonization week?

I think it's primarily, as you said, these high fiber foods, adding the fruits in is important too, because those are a source of fiber, but also polyphenols. We know that polyphenols are beneficial prebiotics for the microbiome, these certain strains as well. For example, we know that polyphenols consumption results in higher levels of acromansia. I guess just to avoid the high fat, high sugar foods.

Let's be specific about each of those. What is it about dietary fat or succrose, fructose and glucose that would be problematic?

It's not about the detrimental effects of them. It's actually about what they don't contain.

I see. Don't eat McDonald's because it's low in fiber, not because it's high in sugar and fat.

Yeah, I guess you're right. The high sugar, high fat foods don't tend to be high in fiber, so I was making an assumption there. But it's really about trying to optimize for high fiber, high polyphenol foods. Anything that doesn't have that is.

Not feeding. Okay. You don't have to avoid eating your meat, even though it's not a source of fiber. You have to make sure you better be eating a lot of fiber and polyphenol. Let's talk about artificial sweeteners for a second. Highly contentious topic.

Yes.

Recently, along with a couple of my colleagues, we put out a very lengthy piece of content to our premium audience, to the newsletter. It's a 15,000-word treaties on all things related to non-nutrative sweeteners and non-sugar sweeteners that are themselves nutritious. It's a pretty broad piece. Without recasting the entire thing, it's really clear that there's something going on with these non-nutritive sweeteners beyond their caloric or non-caloric impact. In other words, we all understand that succrose has its four kilocalories per gram. It's broken down into one-part glucose, one-part fructose. We understand the metabolism of those things, and we understand that if consumed in excess, you have probably some harm beyond just the caloric side, these are the fructose molecule and not the glucose molecule. But it's now also clear that under iso-caloric conditions, high quantities of non-nutritive sweeteners are not entirely benign. I guess I'd start with the question of, what do we know about how the gut addresses these? If for no other reason, because of the fact that these are very foreign molecules in the concentrations we consume them, I mean, we consume glucose and fructose for our entire existence. We're just seeing it in a higher concentration now, but probably not as much of a multiple in concentration as we see aspiratame or succralose.

First of all, I'm going to give all the caveats that you are clearly a far deeper expert in all of this than I am. I haven't spent hardly any time thinking about it.

I know nothing about the impact on the gut, really. Just the observations clinically about what we see in terms of sugar cravings and other repetitive behaviors and metabolic symptoms, which I'm asking, do you think part of that is manifested through the gut? There was that very famous paper in Nature a few years ago which suggested that in mice.

Yeah. Well, first of all, there have been a lot of studies done in mice, and we've already talked about the advantages and disadvantages to getting too fired up about mouse studies, especially in the microbiome. But I think that the data that's out there is conflicting around the impact of these non-nutritious sweeteners on the microbiome. And maybe it's because of what you just pointed out, which is these are not all created equal. And so by lumping them together and doing these studies, that might be causing some of this conflict. I think it's relatively early stage. A lot of studies have done in animals. There are definitely studies which show that they can have a detrimental effect to some of these beneficial microbes. In my mind, the jury is still out because I think we don't understand the complexity of these different sweeteners. We also don't understand, obviously, the complexity of a microbiome and the adaptation of the microbiome to consumption of these sweeteners. And so, yeah, evolutionarily, maybe these things haven't been around for very long, but again, because how fast you replicate.

Because.

Of the rapid evolution. -you replicate your... I mean, there's bacteria that can metabolize small molecule drugs. They've definitely never seen before.

They've never.

Seen, yeah. I think that is going to be the name of the game is to understand how does your microbiome evolve to these and how does it help or hurt you. And how is that linked to metabolic pathway?

My takeaway is all of that and then layered onto that something you said earlier, which is you could take five people who are the same weight and give them the same of Digoxin, and they're going to have five different PA Ks. For those listening who don't understand the term PA K, it refers to the concentration of the drug within their body, in other words, a product of their metabolism. I would say the same is probably true for Aspertame, Sucralo, Sacrin, all of the above, which is I have seen so many cases of people are trying to lose weight, trying to improve their metabolic health, drinking six Diet Cokes a day. They're saying, Look, I'm getting zero calories in here and nothing will budge. Do me a favor, substitute soda water for the Diet Coke for a month. Let's see if it makes a difference. And the world changes. I don't know what to make of that because it's anecdotal and I don't have perfect control over the situation. It's certainly possible that when they started drinking all the Topachico and started ditching the Diet Coke, that they were also doing 10 other things that changed.

I really don't know. But I've seen it enough in both directions where it works and where it doesn't work. I do wonder if there are individual factors where in that individual for whom it becomes a productive change, there's a lack of symbiosis between the evolution of their bacteria in the high aspiratame environment versus the adaptation of another person in the context. My wife drinks Diet Coke. I say that like it's somehow a battle. But she freaking loves Diet Coke. She probably has one every other day. I don't know that that's a high or low dose, but it is.

She's in.

Great shape. Yeah. She's as healthy as a horse. If I drink it, I can't stand the taste of it truthfully. But sometimes I'm so parched and thirsty, I'll drink one. It doesn't seem to cause me any ills. But again, I've seen people, and there's clearly an association between its use.

And otherwise. That would be a super interesting study, I think, to do where you basically take a bunch of individuals, get some baseline data around their microbiome, and then you would put them, you would start them either on a bunch of Diet Coke's or you would start them on a bunch of just soda water and then measure their microbiome over time. And then I do a little washout period or maybe just switch them right over. Do a crossover. Do a crossover and see what happens. Because this is my frustration with a lot of the microbiome studies that are done is they treated the way that we do drug studies, which is you just have cohorts and you're just comparing them to each other. Really, in the microbiome, the person matters. And so crossover designs are going to give you way more information about what's doing what in that person. And then you can start to draw themes about pathways. But that would be an interesting design to do because it could be that at the onset, if there was something about these people who could live off of diet Coke and let's assume people prefer diet Coke over unflavored soda water, if there was something about their starting microbiomes that enabled them to lose weight in that way or be healthy in that way, that could be a solution.

So I'll tell you, it was a very interesting study that was done where they took all these people, they put them all on the same diet, and everybody has experienced this. You don't have to be a physician, you go on a diet, somebody else goes on a diet, one person loses way more weight than the other person. So what they were trying to understand is there's something about the starting microbiome that changes the way people respond to... They just did a regular high fiber diet. And they found that if you start out with higher levels of acromansia, not to keep going back to this strain, but it really is this Keystone strain for a reason. This study, what they showed was that if you had higher starting levels of acromansia, that was associated with all the metrics of responding better to the diet in terms of BMI, hip to waist ratio, A1C, weight, all of these things. Those people did better, and it's correlative. If they had higher starting amounts, they responded better to the diets. I do think there's something about the microbiome that can help you or hurt you as you go through these nutrition changes.

To me, a corollary of what you just said is if your microbiome is suboptimal, which we can define in a number of ways, but let's use one very specific example. If you are completely deficient in or woefully deficient in achromatia, it is harder for you to have a favorable response to a healthy intervention and/or you may be more susceptible to the downside of less healthy intervention. On the one hand, you may be more impacted negatively by something like non-nutritive sweeteners, and you may be less responsive to dietary corrections. Would you agree with that statement?

That's what that data points to. Yeah, that's a hypothesis.

Let's talk about acromatia then. Going back to it. It's the species. There are several strains of it. When you refer to it, are you always talking about the same strain?

Most of the work out there has been done on a particular strain called acromansia mucinophila. There's other acromansias out there, but when people talk about to say the word acromansia, they're really referring to that strain.

Okay. How is it grown? You've already alluded to the fact that it's a hardcore anaerobic. You made some insane statement earlier, which is if even one molecule of oxygen is brought in the presence of its culture, it's dead.

Yeah.

First of all, can you explain to me? I don't remember enough of my microbiology to know why that's the case. I understand why an obligate anaerobic survives without oxygen. I understand that biochemistry. I don't remember the biochemistry of why oxygen is toxic. It must be a free radical thing or something.

Yeah, they're insanely sensitive to it.

Amazing.

Yeah, which is crazy because there's so much oxygen in the air around us. But this is really this compartmentalized body part that we have. But there are these strict anaerobes. You'll see this, especially we talked a little bit about these microbiome therapeutic companies that are developing drugs, almost every single one of those companies has had to develop their own manufacturing plant because all these next-generation strains that we're talking about that are really in the gut microbiome, in the distal colon, they have this same feature, this same issue, which is that you have to create this end-to-end closed system. And so all of us who are developing these next-generation strains and trying to do studies with them, we all ended up having to build our own manufacturing plants in order to solve for this precise problem of the oxygen.

Okay. Were there people out there trying to grow Acromansia without super, super strict controls?

Yeah, for sure. There's a company that is touting pasteurized Acromansia, dead Acromansia as the product that you really want. It's hard to not only grow the strain, but also to maintain its viability on the other side of that as you try to create a product that people can take.

There are lots of products out there that supposedly include acromansia, right?

No. We have a whole game of Whac-A-Mole happening at our company for what we're calling fake Acromansia. These are people who will throw a supplement on Amazon and say, This is Acromansia, and you can test all these things. Most of them just have Lactobacillus or Bifidobacteria frame in them.

They're not even trying to fake it?

Falls advertising.

Just false advertising.

Yeah.

Walk through the process. To grow Acromansia, you have the same vet. But now what do you have to pump into it? Nitrogen? How do you get rid of oxygen? How do you create a no-oxygen environment?

You get rid of oxygen by pumping in a competitor, nitrogen. At some point, we should have you come visit the manufacturing site. We'll get you all gowned up and take you on a tour. Essentially, it starts with a freezer, master stock of the strain. We always go back to the freezer stock because of this genetic component. We have a master stock of the strain, which has been very heavily characterized. We don't want to just keep growing this thing and then go off of that growth and go off of that growth.

I can understand why you would want to start with the Master Stock because of the profound genetic drift. What do you now do? You grow this in a pure nitrogen environment?

You're actually pumping in multiple other gasses. Nitrogen is one of the primary ones. Actually, the mix of gasses matters. That's actually part of the secret sauce of growing the strain. The mix of gasses matter and everything is anaerobic. So in the beginning, when you just have the small stalk and you're doing the smaller cultures, you're literally operating in an anaerobic chamber. So you've got the gloves going to this chamber and you're working in that fashion. And then there is a physical tube that goes from that chamber into an entirely closed then bag, transports into this bag filled with media, and then you're growing it at these larger scales. Everything is an entirely closed lock system, and you're pumping in these other gasses in order to continue to keep oxygen out. Because really, no matter how close the system is, what we found is that oxygen makes its way in. One of the most expensive parts of this process is the pumping in all these other gasses. If you come visit our site, you'll see we just have walls and walls lined with these tanks of these other gasses that are getting pumped in. Then after that, again, everything through lyophilization has to remain anaerobic.

Then once it's freeze dried, now it's in a stable state. I could leave Acromansia powder out on this table for weeks and it would be fine. So it's really just leading up to that freeze-dried state.

Wow. So freeze-dried Acromansia, I guess that makes sense because a capsule presumably still allows for diffusion of oxygen.

Absolutely. And the freeze-dried state, you basically put them into a dormant state, so they're not metabolically active at that moment.

And it's so interesting. So you don't have to keep them in a freezer at home. A fridge is good enough. Even though they had to be cooled to a much lower temperature, presumably to be freeze-dried, they can be warmed to as much as a refrigerator, which is probably what? 38 degrees or something?

Yeah. Well, actually, Akkermansia, the single strain is room temperature stable. You can actually have those pills at room temperature.

That still doesn't permit enough heating.

Yeah.

The body temperature is what's necessary to heat it.

Even then, when you consume Akkermansia, it's the hydration that's actually your big problem once you're in a freeze-dried state. You're basically trying to keep that guy dormant. Once it's freeze-dried, and when you buy our bottle, you'll see we have desiccant packets in there. We have desiccant line bottles when we do our clinical trials. It's all about keeping moisture out. Now, once they're in a freeze-dried state, now, once water enters the game, that's what brings them back to life and then they die because now there's oxygen. Once they're in their capsule form, some people tell me like, I throw out that little packet. I'm like, Don't throw that out because that's what's going to help you keep these guys stable.

Wow. What's that process take in terms of time?

The time varies for each of the different strains, and obviously also the concentration and the number of bottles that you're trying to make.

The people who do this, obviously, this is a high touch industry. They were doing what before this industry? This is something that's done where?

You mean before manufacturing probiotics?

Yeah.

Probiotic manufacturing has been happening around the world for a very long time.

But using this high, intense, anaerobic process.

Actually, we built this thing from scratch. We had PhD scientists and microbiologists who were taking these small-scale methodologies and then trying to figure out how do you grow them at larger scale. In fact, our manufacturing plant is in San Francisco because that's where all of our PhD microbiologists were living. We actually have a pretty young team of people that are coming, almost some of them straight out of their postdocs.

How are you doing the flow cytometry on the anaerobic without introducing oxygen?

The flow cytometer is in an anaerobic chamber.

I mean, I used to do flow cytometry every day. They're huge.

Yeah, we have custom-built anaerobic chambers for all this stuff.

Wow. How does the person go in? Are they just putting their arms in?

Yeah, you just have these gloves. You stick your arms in.

But to prepare for the facts, you're mixing the antibodies, you're puts in around, you're rinsing. You do all that through these little glove chambers.

All of it is done.

Through these gloves.

That's hell on earth. Then there's a little box on the side that you open up, you put your thing in there, you close it, you have to.

Deoxygenate it. It's like a submarine.

It's like a submarine. The double chamber. It is. I think people get pretty adept at using these gloves. I'm terrible at it, but they can do really fine work with them.

Aside from the fact that Acromatia, from an epidemiologic standpoint, always shows up. How long has your company been selling an Acromansia probiotic?

We've only been selling it for a couple of years. I mean, we really spent about a decade doing dev work and R&D work and preclinical and clinical work. We wanted to make sure the thing did what we thought it was going to do.

Are you comfortable saying how much money you spent on that R&D?

A lot. Our company has raised $150 million, and we've only had products in market for about 2-3 years. It was a heavy lift to build a manufacturing plant, do all the R&D work, funding preclinical and clinical work. We fund other PIs to do work with these strains. I think if you want to build a product that has real efficacy behind it, there's a lot of blueprint from pharmaceutical drug industry that we've adopted because the incredible rigor that they have around developing products with efficacy.

Yeah. Your industry is not one that's known for rigor. We'd be putting it mildly.

Yeah. This is a really pivotal moment for our company when we really decided to sell to consumers. We'd always had this idea that the microbiome is really interesting because it's mutable and you could develop things that could help people. There is a lot of evidence suggesting through these fecal microbiome transplants that you can actually help people improve health through microbiome intervention. And that if you followed a drug development, rigorous pathway, you could actually identify the subset of that fecal microbiome transplant that could help people. And if you could figure out how to manufacture them, you could really have a product with efficacy here. Now, how do you bring that to market and how do you choose to commercialize that? We always believed that because these are grass, these are naturally occurring strains, that you had an opportunity to bring this directly to the public that would allow you to really democratize the availability of it. So if we launched pendulum glucose control as a drug, we would only be able to sell it to doctors who would prescribe it to people who had type two diabetes. Anybody who's aging should be thinking about how to help their body metabolize glucose better.

And so we really felt like there was this natural product, things that could deliver advocacy that was measurable, that would be important, and then enabling these products to be available to everybody as opposed to only through a prescription. That meant that the consumer market was really the way to go. Also, a lot of people learned information about health on their own. I mean, Dr. Google is the most famous doctor on Earth, and so we really wanted to bring it directly to consumers. The downside of that, and we've talked about this too, is it's really hard in this particular space to elevate it. I mean, everybody is a marketing genius that's selling a probiotic. There are people who make you feel like what they're selling is super innovative, and you've never seen anything like this before. But when you really look at the ingredients, it's literally the same thing that everybody else has been selling. And because the consumer is not going to go read clinical trials or necessarily compare your ingredients to ingredients and all the other labels or do all that legwork, it becomes really different game to play where you're trying to deliver something that provides meaningful health solutions for people.

But you're also having to play the marketing game of how do I convince people or use proxies to help them understand this works because you're not selling to a bunch of doctors. You're selling to regular people.

Well, even if you are selling to doctors, I mean, quite frankly, it's complicated. I mean, it's not like doctors would necessarily understand this. I struggle to understand this. Let's explain what Acromansia does to control glucose. You've alluded to this a couple of times now. You've alluded to a product called glucose control. Let's go back to the science of this. There's a clear and obvious correlation as to why acromansia is beneficial, but that doesn't explain mechanistically what it's doing. Do we have an insight into why acromansia is something that lowers an individual's blood glucose or response to a glucose load?

Yeah. We'll do a deep dive into acromansia and what we know today, knowing that we're going to learn all kinds of other stuff. But at a very fundamental level, this is primarily through the GLP-1 pathway.

Just to get people excited about that, if you're hearing this and you've heard of OZEMPIC, you've heard of GLP-1. Ozempic is a mimetic of GLP-1.

Yes. What we know about Akkermansia, there's three key things that we know that it does that result in this. The first thing is that it has a surface protein called AMUK 1100 that appears to be able to bind to TLR2 receptors that helps stimulate these L cells. So maybe we'll take one step back, which is that you have these L cells in your gut microbiome, at the lining of your gut microbiome, and L cells are the cells that secrete GLP-1. So a lot of people don't know this. Your microbiome is really the guy who's secreting GLP-1. So you have this beautiful system where you eat food, your microbiome metabolizes it, and right there are these cells that respond to that food by secreting GLP-1. So Akkermansia has this surface protein called AMUK 1100. It also secretes a protein called P9, and P9 binds to these ICAM-2 receptors in the L cells, which are also known to stimulate them to produce GLP-1. And then the third thing that Akkermansia does is it's able to produce a short chain fatty acid called propionate. And propionate is upstream of butyrate. And so there are a bunch of strains which can take propionate, convert it into butyrate.

Butyrate binds to G-protein coupled receptors 42 and 44 also in these L cells and stimulates GLP-1 secretion. And so these are all ways in which Acromansia can stimulate GLP-1 secretion. And by the way, there are only two strains shown to be able to directly stimulate GLP-1. One is achromansia, mucinophila. The other is Clostridium butyricum. And they actually act together because Clostridium butyricum, as its name indicates, is a butyrate producer. And then GLP-1 has multiple benefits. One of which is that you know all this stuff way deeper than I do, but helps your body know that to secrete insulin to help you metabolize the sugars that are in your blood after eating a meal. But it also has this other benefit, which is what is becoming really popular now, which is that induces satiety. It makes you feel full. And it does that in two ways that are not totally understood. But one is that it slows your GI transit, which gives you a feeling of fullness. But the other is that it has some a neurotransmitter mechanism that allows you to feel like you don't have cravings, you're full. It makes it a very powerful small molecule.

And so what we're doing that's pretty distinct from what these small molecule drugs is doing is the natural way. We're giving you the upstream bacteria that's enabling your body to produce GLP-1. The result of that is that your body will produce GLP-1. If you've eaten food, it'll go back down. It'll produce it again after you've eaten food, but it will raise your levels of GLP-1. So you get these benefits of lowered blood glucose, reduced food cravings, lowered A1C, and the body weight impact.

Okay, a lot I want to unpack there, starting with just a quick summary. You eat food, provided you have a lot of acromansia and you're feeding at the right food, a couple of things happen. It sounds like one of them is not at all related to a production or a secretory product, but rather just the surface receptor. I think you said it was the AMAC 1,100 or something.

Like that? Yes. It has a very fancy name. Yeah.

And that surface protein by itself just stimulates L-Cells in the enterocyte. We know that that makes GLP-1.

Yeah. And I think that pathway is probably the least well understood. Short-chain fatty acid and the P-9 protein have had, I think, a lot more work.

Done on that. It secretes P9, it secretes P-9-9. It secretes propionate. Propionate gets converted to butyrate by another bacteria. Which ones?

There is a class of Clostridial strains that will do that secondary conversion, and Clostridium butyricum is the most well studied of those.

Got it. What about Difficil? Is he doing anything good for us there?

Actually, I don't know. We haven't studied whether, and maybe people are afraid to bring that into.

The lab. Yeah. And what does P9 do directly? Stimulates L cells?

Yes. It binds to this ICAM-2 receptor on the L cells and that stimulates them.

Icam receptors are receptors that are usually involved in, God, if my memory serves me correctly, like an immune response, right? Like an inflammatory response?

We have not done studies around that, but for sure, that is an area of heavy interest.

Yeah, by vague recollection, I mean, we're literally going back 25 years is that when a person is, for example, septic, you have all of these ICAM modules that lead to vasodilation, leaky capillaries, secretion of monocytes out into the peripheral tissue as macrophages. But it sounds like this is a distinct process here.

Maybe I should say too, I don't know how much of this is tied to that process, but not only does Acromycin stimulate these L cells to produce GLP-1, but Acromycin has another role, which maybe is very much tied to this, which is that it helps regulate the mucin layer. That mucin layer turns out to be super important for if it gets too thin or if it's too thick, it's a real so-called leaky gut or GI issues that could become a result of not having enough acromansia is really that you have too thin of a mucin layer. You start to get the ability for pathogens to infiltrate and then also obviously for these molecules to secrete out.

Then tell me what's the fate of the butyrate made of the propionate?

It binds to these G-protein-coupled receptors, and that's also what stimulates the L-cells to then go release GLP-1.

Got it.

There are multiple these small molecules that are the signaling molecules for these receptors on the L-cells.

All roads point to more acromatia, more GLP-1. What differs from someone taking Ozempic is they just have a mega high dose of GLP-1 all the time. And what you're talking about here is, as you said, a waxing and waning dose of GLP-1 that is more physiologic because it comes with your meal.

That's right.

So one would not expect this type of intervention to produce the amount of weight loss you would see with carpet bombing somebody with GLP-1.

Absolutely not. One of the other big differences is that the GLP-1s are injected. It's going right into the bloodstream, whereas it's a microbiome effect. To your point, you get the waxing and weaning, more physiologically relevant. But it's not going to be the same as just hammering a bunch of GLP-1 straight into the bloodstream all the time, all day, all night.

Now let's talk about the data. What was the first study that demonstrated that acromatosis could play a role from an intervention perspective from impacting metabolism vis-a-vis blood sugar?

Yeah. Some of the earliest studies are really done. I mean, this, for example, was discovered by Dr. Lee Kaplan over at MCH, and he's a bariatric surgeon. And so his initial interest was what's happening to the microbiome? We do this bariatric surgery and came to really be one of the first people to really look at these microbes and discovered that acromatia appeared to be associated with or inversely associated with obesity, and then started doing these in vitro studies to figure out what is it doing. The work really has to go credited back to him in the early 2,000.

I want to point out one of the observations that came from his work, which is a really remarkable observation, which is at least as far as the Rue and Y gastric bypass, which is a real organization, a reorganization of the plumbing of a person's gut, that you took a person who was obese with type two diabetes and you do a Rue and Y gastric bypass on them. Within days of surgery, their glycemic control improves even before they've lost weight. This is a very important point because nobody would find it that surprising if a person after a gastric bypass loses 100 pounds and their diabetes goes away. That would be a Oh, shucks. Of course, moment. It's why does their diabetes resolve in the days following surgery when the real weight loss hasn't started? There have been lots of to this. One could be the fasting. You're not eating anything a day before surgery or a day or two after surgery. That's a pretty significant fast that depletes all of the glycogen in the muscles and much of the liver glycogen. Is that enough to kickstart them? But it's hard to ignore that you're also completely changing their gut biome by re-plumbing their GI system.

Can you say more? Because I'm not familiar with those data in terms of what they saw as far as pre and post surgical gut biome.

I'm also not an expert in these surgeries. They have been a source. A lot of publications have used them to try to understand what are the key gut microbes out there. I don't think it answers this question of why do you see such an immediate response sometimes hours after the surgery? Just thinking about the time length of these different pathways to have effect is most likely something hormonal. But I don't think we have the answer to that.

It's hard to imagine it's not something related to GLP-1.

Yeah. I don't think we have the answer to that specific question, but definitely that was where Acromansu was born out of. They do these gastric bypass surgeries in... There have been a bunch of animal models. This was some of the very early work, really just trying to lay the foundation for the fact that your microbiome could play a role in weight loss or weight gain. And so they would take these microbiomes before and after the surgeries, and then they would put them into these mouse models, and they would show that you could change the of the mouse. It's been really hard in the field to take that data, that fecal microbiome data where you feel like, Oh, there's a glimmer of hope here, and to distill it down to what are the things that are actually doing that? That's the big challenge of the field. We're not there. When you say like, Oh, what did they find? Well, they found that the whole gmish could change things, but we don't really know what in there is doing that.

Yeah. One hypothesis is that acromansia is doing part of that, at least. Yes. The easiest way to test that would be to give people acromansia. Okay, so you guys did an experiment like that. Who did you do that in collaboration with?

I'll point to the clinical trial that's published in BMJ because I think that was probably the most rigorous trial that we've done. Then I'll talk about the downside of running too many trials and things that involve humans. But in that trial, we didn't just have acromansia. We actually had it in combination with four other strains. The idea here is, as I said, acromansia can produce propionate, but without the help of another strain, it can't produce butyrate. And so we had this idea of let's include primary and secondary fermenters. Let's not make acromance the only primary fermenter. Let's add some more in there. And really thinking about what is the team members that you'd want in here that could metabolize fiber into butyrate, trying to optimize her butyrate production. At the time that we did the study, nobody even knew what P-9 was or AMUK 1,100. We just knew that we thought it was only playing a role in the mues and regulation. We didn't even really know that it was able to stimulate GLP-1 even at that time. We really thought it was these butyrate producers that are doing that.

Which are the Clostridial strains?

Yeah, which are these Clostridial strains. Then also this bifidobacterium infantic strain, which is also a primary fermenter. We basically did a placebo-controlled, double-blinded, randomized trial where we had three arms. One was placebo. One was this full formulation of all five team members, and then the third one was a subset that did not include acromansia. And these were in people with type two diabetes. Initially, we wanted it to be people who just had type two diabetes but weren't on any medication. It turns out that's almost impossible to find. Yeah, it's a.

Hard study.

To find. Yes. So then we said, okay, we'll have it with people on Metformin. Anyway, a lot of people are on Metformin. So if your thing is going to benefit people, it ought to work on top of Metformin. Recruiting into clinical trials is hard. It takes a long time. We are a startup company. We're like, we're going to run out of money if we don't expand this out. So then we started including people on sulfonylureas. Finally, we get all the people in trial. It's still a pilot trial, 76 people across these three different arms. What we found is that compared to the placebo group, the people who were on the full five-strain formulation over a 90-day period saw their A1C go down by 0.6 and their blood glucose spikes go down by 34 %.

Measure how?

That was done using a pretty old-school oral glucose tolerance test. They literally came into the clinic, they got their sugar, and then they just had blood drawn every 15 minutes for two hours. The reason we did that rather than a CGM was because we wanted to use all the gold standard traditional methodologies. We don't want anybody to think that the data was weird.

So hemoglobin A1C came down by 0.3 %, which is a pretty big drop.

0.6 %.

Compared to placebo. Compared to placebo in 90 days and peak glucose level fell by about a third you said?

Area under the curve. So the entirety, the area under the curve.

Okay, fell by about a third? Yeah. Got it. That was placebo to five strains, acromansia plus four strains to do the conversion of propionate into butyrate.

We also have strains that do the redundant function of acromansia, which is they'll do that primary fiber into the propionate or acetate. I see. Okay.

Then how did the group absent Acromansia do?

They had some efficacy, but it wasn't like the five-strain formulation. My co-founder and biostatistician would say that was not statistically significant and the data is published so anybody can go look at it. I would say it looked like it was in between the placebo and the five-strain formulation.

But it could have been very underpowered, and that's why you didn't see a difference.

It also could have been very underpowered. Yeah.

This was a thinking about this now, not as a scientist, but as an entrepreneur. This was a do or die moment.

There's going to be a binary outcome here. Either we were going to be a company on the other side of this, or we were probably going to close up shop because it was all in on this trial.

What year was that experiment done?

This was maybe 2018. We found it in 2012. But all the stuff leading up to this was we had identified the strains, we had to figure out how to.

Manufacture them. Six years of basic science.

Six years of basic science leading up to this trial. I was already starting to think about, it's very rare that your first clinical trial out of the gates works. This is why we did the three strain and the five strain.

Yeah. Why didn't you just do the placebo versus the five strain?

Because acromansia was so hard.

To grow. Oh, so you were hoping to see that the four strain would be great, and you wouldn't have to grow acromansia.

Yeah, we were hoping we didn't need the Diva in there. It turns out and we did.

So what's interesting scientifically, but would have added too much overhead to the study, and maybe it's irrelevant if the other four are not that expensive to grow, is with acromansia alone, how would that have performed relative to the five strain pot?

Yeah, of course, if we had been able to do that subsequently. But I will say that lots of people have been doing work with acromansia solo. I put Vegas odds down that by itself, it's probably not as impactful as the formulation.

Because you don't have the assistance in converting propionate to butyrate. Exactly. Sorry, dumb question. I think you already addressed this, and I've forgotten already. Butyrate is stable. It's a short-change fatty acid. Why aren't we just all mainlining butyrate?

There have been a ton of preclinical and, of course, in vitro work showing the massive benefits of butyrate across a bunch of different states, including metabolism. Yet none of them is translated into humans. Even when we try to do these enomas, these clinical trials, they've never looked the way they did in the animal studies. The animal models are like blow your mind, glow you out of the water, and the human trials really don't show that. And I think for this particular pathway that we're talking about here, it's a localization problem. So earlier I mentioned that butyrate is a source of energy for all these colonic cells. The issue is when you're delivering butyrate all along that track as this butyrate is basically being absorbed by all these different cells. And where you need it to get to for this pathway is to the gut lining where the G-protein-coupled receptor is sitting, bind to that G-protein-coupled receptor.

It's not that acromansia just makes butyrate. It's that it delivers it to the place where it needs.

To be. Exactly. Literally, the physical proximity is what's enabling this to work.

Let's go back to actually something you said a moment ago, which is this trial was done in people with type 2 diabetes. Average hemoglobin A1C coming in was what, roughly? These are early diabetics. They're in the sevens?

Yeah, they were in the sevens between 7.5 and 8.2, I think was the range for all the groups. I have to go back and double-check that.

There any sense? I know you didn't do this in the study because you said it was only 90 days, but is there any plan to redo this to find out what happens if they had stayed on for a year? In other words, could you take someone with type 2 diabetes and actually knock 1.2 % off the hemoglobin A1C, which for many people would now take them straight out of the diabetic range?

Yeah. We have multiple ongoing studies. Maybe this will get back to the personal story of the entrepreneurship, which is that on the heels of this study, we were like, Holy shit, we made something that works. Now we got to figure out how to commercialize it. But before we do that, we should probably replicate the study, or maybe we should do them in parallel.

By the way, did you get any pushback from your investors on that?

Doing another study?

Yeah, because if you're wearing your scientific hat, that's the obvious thing to do because scientists always want to replicate things and make sure and add another question. If you're an investor, you might be like, Are you crazy? We just got the answer. It's the best possible answer. Don't ever ask another question. There's a reason virtually no supplement companies will ever, ever, ever do a clinical trial. They don't want to know the answer.

It's true. Well, there's two things. First of all, we have been super fortunate in having investors that believe in the underlying premise of our company. And the underlying premise of the company is that the supplement space is riddled with tons of products that don't really do anything. And therefore, it's very hard for anybody to own market share. It's actually a highly fragmented space. Sometimes you get a player that comes in, they take market share because they've done something fancy on the marketing side, but then somebody else comes in and takes their market share. You just see this game happening. Our underlying premise was if you could deliver something that actually helped people in measurable ways, you could take that whole market over because now you've created something that works. Well, in order to know if it.

Works- Yeah, you have to have a study and you have to make a claim. You can now make a claim with a product. That's a very big deal. Are there other probiotic companies out there that can make claims?

No, there's a claim around lowering A1C and blood glucose spikes.

You own that claim?

Yes. We are the only ones that make that. Well, somebody else could do a study and make that.

Claim too, but they haven't. Yeah, but currently nobody.

Else can make it. Nobody else does. The other reason I think our investors got behind it is, first of all, they understand that we're trying to build products that can change lives. We have investors that have gotten behind game-changing category creating products in the world. So Apple and these sorts of companies that maybe didn't have a predecessor to them.

That's a very different risk tolerance than biotechnology. Biotech is the graveyard of some great investors.

It's true. I don't know what to say. They're behind this mission that the thing has to work, and so therefore you've got to have these studies that work. I will say, though, I have learned by having products in market and enabling people to run their own studies on themselves, especially in the context where nutrition and your ecosystem, your microbiome are still pretty much a black box to the scientific community. We've been able to garner so much data from our customers who are just sharing this stuff voluntarily with us in order to be able to do better and better product development. So I'm actually now a believer that the clinical trial is important because it gives you a very controlled environment to know whether there's a there, there. But unless you can change behavior in the real world and have people actually experience benefit that they can come back and tell you about, you haven't made a product that's very interesting at all. I think that that part is important. The other part to this story that's important on our strategy is that because we have this clinical data and because a consumer may not be able to go read a paper, but a doctor can, that we go through healthcare professionals, we go directly to them, but then we also go through them to help create credibility or knowledge for consumers.

And so if you want to convince a bunch of doctors that the thing you have works, they're going to see more than a pilot. And they're also going to want to see it run by somebody that's not yourself. And so we immediately launched these studies and then COVID hit. And every study, one by one, got brought to its knees because people with diabetes are more prone to COVID complications that came out a little bit later on. But people were getting COVID. And then you're like, Well, I don't know what this COVID is doing to the microbiome. And then people were totally not adherent to any protocols. They couldn't come into clinic. So one by one, we lost a lot of money on trials that we were trying to do follow-ups on. And then we just decided, my chief medical officer said, All right, we have to wait until everything is cleared out before we even start another clinical trial because we're not going to lose another dollar. And so literally, just in this last year, 2023, is when we felt like there was enough of a handle on this that we could actually start doing clinical trials.

But we've done it in a different way, which is now we've built up some momentum. It's all third-party PIs and academic and clinical institutions that we're just giving free product to run these trials. We'll get that data. I do think it's important if you want to get the medical community behind you, and I think that'll be a differentiator.

That means they have to apply for their own grants to fund the study. They're just having a significant part of the cost taken out of it, which is the drug.

Yeah.

But that's good. There's interest. There are PIs out there who are like, Yeah, I would happily go and seek the grants to do this study, especially given that I can mitigate the drug cost.

Yeah. The government is putting out NIH grants. There's quite a few of them centered around microbiome interventions.

How many such trials are underway right now?

Right now we have about a dozen around the world that are happening. Not all of them are for type 2 diabetes. We actually have several where investigators have their own hypothesis about what these strains are doing. For example, we talked about the gut-brain axis. We have an investigator that's been funded by the Milken Institute that is looking at the role of acromansia in bipolar disorder.

That's amazing if you really think about it. You've got basically a dozen PIs around the world on the basis of one relatively small pilot study that was very well done, published in a very reputable journal. That alone has generated that much interest that they're finally willing to study a commercial product.

Well, I don't think it was just that study. There's like over 3,000 publications on acromansia that people around the world have been publishing on, mostly academics.

But those studies weren't intervention studies.

No, a lot of them are correlative studies.

Right. But that's my point. What you did is quite unique in that you finally bridged a gap, which was correlate, correlate, correlate. But until you can test an intervention under blinded, randomized conditions, you can't know if there's an associative link, and you've at least suggested that.

Absolutely. I think that you're right. The fact that we can make it and that it was able to have this efficacy does give people some belief that, okay, well, if I want to go test an intervention, these are the people that have.

Made it. They could never invest in the CapEx to go and make it.

No, I mean, our manufacturing plant, it costs us $10 million to make that plant. Yeah.

Okay, so how many products do you sell today? Pendulum sells the product that was tested in that trial. Is that the product called glucose control?

Yes. So pendulum glucose control is marketed for people with type 2 diabetes, lowers A1C, lowers blood glucose spikes, and it is the exact formulation that was in that clinical trial.

And do you still use that same quality control metric of actually doing flow cytometry on those products?

Yeah.

I take glucose control. If I looked at that bottle, which I can't say I've looked at it and remember, it doesn't say anything about CFUs on.

The back. It says AFU, active fraction unit.

What does it report it in?

It's in the number of cells that appeared in that active fraction. It's still going to look like, I think it's like 10 to the 8:00 or 10 to the 9 for most of the strains. Got it. Okay.

What other products do you sell?

We released pendulum glucose control. I would say one of the hallmarks of our business that we've been very proud of is the repeat business. We get a lot of... Once people try it, they really stick with the product. We actually got a lot of feedback from people. We were getting amazing reports of, Oh, my A1C is lowered, my glucose spikes are lowered. When we launched our first version of glucose control, we also offered people free A1C testing every 90 days and a nutrition coach, which we had a team of registered dietitians. We were like, This is going to be a solution. We're going to give you your nutrition coaching. We're going to give you the test that's going to tell you if.

It's working. How did you offer that for free? You didn't want to make money, I'm guessing.

We don't offer it now. Somebody even said, Oh, my gosh, everyone should go buy this product right now because these are non-scalable. So at some point, they're not going to offer these things. I laughed when I saw that. I was like, Oh, you don't know. But then eventually I realized, though, that's true. I think that it's still important for us to offer nutrition information. It just doesn't necessarily have to come through a registered dietitian and a personalized one-on-coaching. And maybe AI will help us get there faster than we would otherwise.

And what other things did you hear from customers? Did you see in the real world comparable reductions in hemoglobin A1C?

It was all higher. The reports we got back were higher A1C drops, higher blood glucose drops.

Do you have a sense of how much selection bias was going into that? Were there a good number of people saying, What the hell? It didn't get better?

No, actually. Moreover, I would say you might say, Well, gee, those people just didn't tell you, but you can see it in the numbers. You can see it in the return purchase rate. Pendulum glucose control is $165 a month. That's a real out-of-pocket expense.

I don't really know the space that well, but how expensive are the top selling probiotics?

A normal probiotic is 20 bucks a month. A premium high end probiotic is 49 bucks a month. So 165 is way out there.

I think you've explained why it has to be that expensive. I won't ask you what your margin is on it, but I'm guessing it's not that high given your manufacturing process.

Well, what's even worse is that you're teasing me about offering this test and this nutrition coaching and how did you do that. We were not only losing because of those, but we were actually losing on every bottle we sold because it was costing us more to make it than the 165 we were selling for. It's all a hot mess out of the gates. But I think what we believed was that we knew that as we scaled, a lot of those costs would go down. Just like everything else, if you're buying 10 bottles versus 10,000 bottles, the same bottle costs you a lot less.

Can you currently make a bottle for less than whatever that cost is?

We now do make it for less than what we're pricing it. But we also have to ship it cold. There's also that transport.

Yeah, that's an important point. It arrives, you get three bottles, which is a three-month supply. It arrives in an ice pack in a big box, just like if you were to or something like a PCSK9 inhibitor, same thing. Shows up cold, you got to put it in the fridge right away, can't travel with it. There's some logistics challenges associated with these things.

There is definitely complaints. But I think what we wanted to do is to give the product the best chance of success by giving people these tools to be able to help them measure. And then also a lot of people don't even know what a high fiber diet is supposed to look like. Just giving those tools to people. So we got all this great feedback. If the efficacy was higher than we'd even seen in the trial. But we also got a lot of complaints. Hey, man, how is this $165 a month? Why isn't my insurance covering it? Why does it have to be cold? I can't travel with it. What am I supposed to do in the time that I'm traveling? I don't have type two diabetes. We took all that and said, Well, let's just run some marketing tests on a lower dose version. That had huge uptake. And so we launched a product this year called Metabolic Daily. And it's literally the same thing as glucose control, but just at a lower dose that allows us to get down to this price point of $49 a month, which feels a lot more manageable for people when they're thinking about, especially if you don't have type two diabetes, but you want to help your body metabolize sugars and carbs better and things like that.

That product still has all five strains in it?

That product still has all five strains in.

It, yeah. It needs to be refrigerated?

All of our products really should be refrigerated. That does maintain the viability for longer. Those two products. Then we also released the single strains that are components of these products. We had a lot of people who came back to us and said, Hey, I'm buying your pendulum glucose control, but all I really want is acromansia. I just want that one strain. We're like, Who the hell knows what Acromansia is? This is just some really educated people coming to us, and it's not a real market. So we did a market test. We made 1,000 bottles of acromansia. We literally just called it acromansia, and we put on there for gut health. We made no claims about it. People were coming to us for a wide variety of reasons of why they wanted acromansia, from metabolism issues to GI issues to neurological disorders. We were like, we'll just sell it as just Acromatia. That's the value prop. We made 1,000 bottles in less than 10 days every bottle was gone.

And that's an expensive product.

Because it's Acromansia, right? Acromansia is one of the hardest guys for us to make. It's the expensive one to make, yeah. And so that is a product that's one of our best sellers is just pure Acromansia. Actually, a lot of practitioners use it because let's say they ran a gut microbiome test to person's low in Acromansia, they just want to give them the minimal viable product to boost this.

Strain backup. But we're really on the outskirts of knowledge here. We really don't know what it's doing by itself.

Well, we know that it's secreting some of these proteins.

But in terms of outcomes, that's more of a stretch, right?

At this time? I would say that what we've learned from having it in the market is that people are getting a variety of different measurable impact from it. One of the most interesting things that we learned is that we had a bunch of people telling us, actually, the reason why I stay on it is because I don't have any more sugar cravings. I went to the Christmas party and didn't eat a single cookie, and I'm usually the guy sitting at the table eating all the cookies. We started to see this theme of sugar cravings. As we talked about earlier, one of the hallmarks of GLP-1 is improvement in satiety. We just did a little pilot study ourselves. We said, go on this product for 90 days and do this diagnostic test on cravings.

Was it a blinded study? Was there a.

Placebo arm? No. This is a pilot. This is just a proof of concept. What we see is that people have a significant reduction in these food cravings. What that's now being parlayed into is funding a clinical trial to look at that. This is where I think the consumer space is a really interesting place to play because rather than going all in with millions of dollars on a clinical trial based on a hypothesis in your head, you now can put products out there to people, hear back what they're experiencing.

Yeah, generate hypotheses.

From customer data. Get prove concept data, and then you feel like, okay, a priori, I have some sense this thing is going to work. You could even get really sophisticated and say, okay, well, maybe I'd like to know what are people starting microbiomes or what are their diet or what are their lifestyles or the demographics to try to understand who might this thing have the most efficacy for.

Now, you also haven't done the study, I'm guessing where for those... Look at your pilot study or your 76-person study. It would be very interesting if you go back in time, if you had the funding to look at what the constitution of the gut biome was prior to the study, after the study, and if that predicted response. In other words, is the greater the deficit of acromansia at the outset of the study, a predictor of a greater response upon normalization. Because even though the average hemoglobin A1C came down by 0.6, there must have been people for whom it came down by over a %, and people for whom it only came down by 0.2. So do you have a sense of what that relationship is?

Yeah. Well, actually, in that trial, we got four stool samples from people during the trial. Oh, you did? Okay. So one baseline, I think it was like 30 days in after the 90-day mark, and then we did a washout period. So you went for a month without taking anything, and then we got a stool sample. And I'll tell you this, we racked our brains about how are we going to get people to provide four stool samples? I mean, this is like a real ask. We wanted the whole sample. So we literally like these cool whip. That's what we gave to people. They had to literally shit in a bucket, put it in their own freezer until they could get to the clinic and everything had to stay frozen. And so we had 100 % compliance. We had people who dropped out of the study who were still sending us their shit. I mean, it was amazing how much sample people were willing to share.

By the way, I remember my brother got back from, I don't know, he was somewhere in Africa or something, and he had some awful GI bug. Eventually, he had to collect a stool sample. I was looking at his fridge and there was this bag in there and I was like, What's that? He's like, It's just some shit. I was like, No, seriously, what's that? He goes, It's just some shit. I was like, Dude, if you don't want to tell me, that's fine. But he's like, No, that's what.

It is. That's literally what it is. Yeah, exactly. People are sending us shit. The reason is because, as I said, we were an early-stage startup. I was like, The train, sir, this clinical trial isn't going to work. I got to know we have to have this microbiome information so that in my head, I thought, for sure, if this thing.

Doesn't work- I want to at least know it didn't work.

Because- Exactly. Did it get delivered or not? We really were wanting to see the presence and absence of our strains. But to your point, we also really want to understand, could you do this predictive modeling of who would be better or worse responders? We couldn't. It turns out that could be that the end is too small. It could be that we don't know enough about these individuals. But the microbiome alone or even the levels of acromancy alone are not enough of a predictor.

They're not enough of a predictor of response. Tell me this. Did anybody who took acromansia have a significant increase from pre-study to post-study during the 90 days? Then what was the fall-off from 90 days through the washout?

Every participant in the study showed an increase in all the strains. That was very rewarding because we invest a lot in the encapsulation to get the thing delivered.

That was a great proof of concept. We actually managed to deliver something that in theory is undelivered. Exactly.

Deliver the goods. Then the washout period was 30 days. After the 90 days, there was a 30-day washout period. Most people lost the strains after that period. But there were about 15-20 % of participants who were able to maintain their acromansia levels even after not taking the pills anymore.

I was exactly about to ask you that question. Is there any chance that those are the people who just consumed the best diets and ate the most fiber after?

This is another decision that maybe in retrospect. We didn't do diet logs. The reason we didn't do diet logs is because I'm told that people are lies. When you do diet logs in clinical trials, what you get back is all the days somebody was good, and then big gaps of missing days where they ate something terrible or did something bad.

You can almost think of doing a diet log here where the only thing you're asking them to log is fiber. You give them a really clear sense of fiber, and it's just all we're doing is counting grams of fiber per day. Take a picture of everything you eat that is one of these things on this laminated card. Every time you eat a carrot or a celery or this or an orange, just take a freaking picture of it and tell me how much of it you ate and that's it. I don't care how many calories, how many diet Coke. I don't care about anything. Just eat all the cookies you want if you want. I just want.

To know this. Yeah, I wish I'd met you when we were doing the trial design. We didn't do that. What we did was we asked people, What are you? Are you an omnivore? Are you a vegetarian? We have all that. There's what it appear to be a correlation with that. Then we ask people, Please don't change your diet. The whole point of this study is we don't want people to have to undergo a behavioral change in order to see an improvement. It should be just the microbiome intervention. But of course, we don't know if people change their diets. I think what we want to tackle now is a much more direct thing, which is to say that you can deliver people meals and they don't have to eat the meal every day. But basically, if you say part of the trial is you're going to be on the product, and then we're going to deliver you basically a high fiber meal. We want you to have it for lunch three days in the week, and then you have your control group where they just get the pills. I think by deliberately making sure that people are getting this added fiber, you can compare whether that fiber is helpful.

It almost feels like a no-brainer that people who will have higher fiber food will do better. But youwe'll measure their microbiomes and we'll see whether the strains are actually even higher for them too.

The next two years really is an interesting time for your company. But more importantly, I think just our scientific understanding of what's going on on the basis of a product your company has figured out to make. If nothing else, you guys have figured out how to make something from a manufacturing process that no one else was going to figure out. There's no university that could ever have figured this out because they wouldn't have been able to put the resources in it. No one was going to spend 100 million to figure out how to make an obligate anaerobic when the maximum NIH grant is $480,000 a year. It's not going to happen. Now it enables a bunch of people to ask these questions about what's the impact on depression? What's the impact on ADHD? What's the impact on obesity, type 2 diabetes? It's super fascinating. In many ways, it feels like the bridge between the diet and the drug because we already have a really good sense of like... It's not like it's rocket science from an efficacy standpoint to get somebody to lose weight by changing their diet. The effectiveness is the problem. It's too hard to implement for most people.

At the other end of the spectrum, we clearly know now how to do it with drugs, and that's really changed. I think it really started in 2014 when liraglutide came out, but clearly, semaglutide was the game-changer. That's three years ago was when we saw the pivot from semaglutide as just a diabetic drug to an obesity drug. It's exciting for me to just watch this and, frankly, even look at other questions that haven't been answered yet. The dose response is more better. Do we know anything about how much is too much? And what's the maximal dose, the minimum effective dose, and the median effective dose? There's all this other stuff that I don't know. It'll be five years and we'll be sitting here and hopefully having a more interesting discussion.

Absolutely. I think the dosing is interesting one too, because the right question is, what is the colonization that's happening? Because it's not just about what you're delivering, it's about what's colonizing. It's different from person to person. I think if we could crack that nut on the dose to colonization ratio and in what context that's better, you start to see improvements. But absolutely, it is the gap between nutrition and then these downstream small molecules. The microbiome has been a black box, and now we've got some tools here. We're just at the beginnings of it. But I think for us, the big name of the game is how do we get these products into as many people's hands and as many investigator's hands as we can to just create more and more data around what is your starting microbiome? What is your lifestyle? How are these things tied together and what are the health outcomes that can really come from a different microbiome?

Are there any other bacteria out there as specific strains or even species that you think are going to be worth investigating based on the literature today? We've talked a lot about acromancy and all the reasons why you had so much data to stand on the shoulders of to go down that rabbit hole. Are there others out there that you think offer promise and that maybe in five years, there are many other strains out there that people are taking. You might have in your fridge a bottle of Acromansia strain two, strain three, strain four?

Absolutely.

I assume you're not going to say what they are. I'm guessing that's somewhat proprietary.

At this point. Absolutely. There are other, I think, of these key strains that I'll just allude to the link between our microbiome and our immune response is super interesting. There are other strains.

We'll stay tuned.

All right, thanks, Colleen. Thank you so much for having me.

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