] infection as a disease in general. I would characterize this as the original microbiome disease. What I mean by that is there’s a very solid and clear link between the microbiome and 

 infection. It’s been known since roughly 1978 that 

 infections were the most prominent of a class of infection called antibiotic-associated diarrhea. It is what it sounds like. Antibiotics are administered to the patient and the patient develops diarrhea as an adverse effect. Now why is that? We think about the microbiome as being protection in our guts from invasion by host pathogens, 

An analogy I like to use is to think about a really diverse and rich flower garden. We all can see seeds floating on the wind; some of them will occasionally take root, but if you were to clear cut that garden, those roots would have access to the soil. They’d spring up and take over the garden really quickly, and that’s what we see in the gut. The normal microbiome in a healthy gut is like that diverse garden, it has many different colors, flavors, and functions that help the gut function well. But when you damage that through the use of antibiotics for another reason, then you reduce that ability to resist colonization by 

 very often then will come in and take over, proliferate, and cause serious infection.

 itself is a gram positive, anaerobic, spore-forming bacteria. What does all that mean? It’s spore forming, meaning that 

 has the ability to form a very small spore, something like a seed that’s protected from the elements. Because of that, it easily spreads. It can spread through contact and fecal material, and that’s how it gets dispersed throughout a hospital system, which is one of the primary risk areas for 

 is anaerobic. That means that it needs to have an absence of oxygen to grow, and that’s important for its lifecycle. It doesn’t just grow anywhere, it only grows in the absence of oxygen with the exception of when it gets to be that sporiform.

 is able to proliferate in that damaged microbiome, or the damaged garden of the gut, it actually grows, proliferates, and takes over the environment. You had asked about virulence factors—at that point it expresses its primary virulence factors, which are called toxins A and B. Both toxins A and B will cause changes in the shape and function of intestinal epithelial cells. It damages the cells and causes loose injunctions in the intestinal epithelium, which leads to a variety of symptoms, ranging from mild to severe. Milder symptoms are things like diarrhea, bloating, and cramping. More serious symptoms are severe cramping, severe diarrhea, severe intestinal inflammation, also known as toxic megacolon. It can be life threatening.

It’s a serious and very concerning disease, but it also has a lot of quality-of-life issues, particularly when it becomes a recurrent disease. Think about the challenges of being tethered to home. Patients are undergoing an awful lot of symptoms. It affects the quality of life, their ability to function at work and socially. It’s a real challenge for patients, and it’s a real unmet need that we’ve tried to address very early on.

Another aspect that’s very important with 

 infections is the likelihood for recurrence. Many times, roughly 25% of patients who get their first, or primary, 

 infection, are prone to develop a new recurrence of that infection. The reason for that is the standard of care for treating a 

 infection is an antibiotic, which although it kills 

, the bacterium, it also very often damages the gut garden in which 

 has sprung up. Therefore, you’ve killed the weeds, but you still don’t have any flowers there to protect the garden against reseeding and reproliferation of those weeds. When we started Rebiotix Inc in 2011, it was with the recognition that something is needed to replenish that healthy microbiome of the gut. That’s the type of investigational therapeutics that we sought to develop.

Once patients advance to the stage of having a single recurrence, when they’re treated with an antibiotic, they’re very prone to have an additional recurrence. Patients who have a single recurrence will then have a much higher likelihood of having additional recurrences. Fifty percent or more of those patients will develop an additional, and an additional, and an additional 

 infection. It’s a challenge, particularly for the patients and definitely for the health care system as well.

The biggest risk factor is antibiotic, particularly broad spectrum antibiotics, and even more so, antibiotics that inhibit gram positive or anaerobic bacteria. Those are the ones where you’re going to see the highest risk factor. Health care setting is another risk factor, as well as long-term care setting, age, and the immunocompromised. Working downward in priority, all these are certainly risk factors for developing 

 infections, and in many cases, the more risk factors there are, the more likely the patients are to recur as well as contract that first infection.

Clostridioides Difficile Pathogenesis & Manifestation

 What the guidelines recommend for a first episode of 

] is an antibiotic called vancomycin. That’s the most commonly prescribed antibiotic for the first episode. A newer antibiotic approved in 2011, known as fidaxomicin, is also indicated, particularly for the severe first infection. If neither of those are available, then metronidazole, an older 

 antibiotic, would be indicated per guidelines. Once a patient advances to that first recurrence, then the recommendation switches to an additional course of vancomycin, or what’s called a vancomycin taper, which is an interesting concept. It means that we’re going to go in there, treat the patient with vancomycin for several days, and then slowly decrease the vancomycin dosage over several days and space it out. The idea being that we know vancomycin can cause some harm to the microbiome, but if we kill the 

 and then we taper back vancomycin in a way that will keep 

 at bay while allowing some of the microbiome to grow back, then perhaps we can avoid recurrence.

Another antibiotic course that’s recommended for first recurrent 

 is fidaxomicin. It’s generally thought to be more microbiome sparing than vancomycin. That’s why it’s indicated in the first recurrent patients. Once patients have progressed past that first recurrence to multiple recurrent 

, that means that they’re on their second or later recurrence, or they’ve had at least 3 episodes total, then the recommendation goes to strictly a vancomycin taper, or fidaxomicin, or a combination thereof.

The newer guidelines from 2017 also recommend consideration of what’s called a fecal microbiota transplant [FMT]. They do recognize that this is not an approved therapy, but they recognize that there are a lot of data in the literature to suggest it may work. A fecal transplant would be very much what it sounds like. A donor comes to the hospital, they provide a stool sample that’s homogenized into what’s called an FMT, and administered to the patient, usually by colonoscopy. This is done after antibiotic treatment. The idea being, can you add bacteria back into the patient that restores a microbiome that could resist 

 recurrence? It’s important to note that the guidelines recommend that at least 2 courses of antibiotic be considered, and they recognize that this is not yet an FDA-approved treatment.

Managing Recurrent Clostridium Difficile Infections

 I mentioned the concept of fecal transplant. That’s a procedure that’s conducted in the hospital. RBX2660 is an investigational live biotherapeutic. What that means is it’s developed under the guidelines for the FDA, called Investigational New Drug guidelines, which is aiming toward a potentially approvable product that could be marketed as a typical drug. It is a broad consortium investigational live biotherapeutic. That means that in making RBX2660, we try to capture as much of the healthy microbiome, put that in the investigational product, and administer it to patients.

It’s also what we call standardized and stabilized. Those are very important concepts. Standardized means there are good manufacturing practices that allow us to ensure consistency, dose to dose consistency. When a patient gets a treatment with RBX2660, it would be the same no matter when or where they’re getting it. It’s also stabilized, which means that it has a shelf life, the same as any investigational drug would have. You can store it, you can trace it, you can track where it’s shipped, and you can expect that it should meet quality control guidelines to deliver to the patient a consistent dose. That was the investigational product in the trials that we presented about.

The specific trial is called PUNCHCD2. This was a trial conducted a few years ago. It was the first randomized, double-blind, placebo-controlled trial of this type of investigational live biotherapeutic. It was a really exciting trial at the time. We still find it quite exciting and groundbreaking. The question that trial asked was, what is the efficacy of RBX2660 to prevent or reduce recurrence of 

] at 8 weeks after treatment compared to a placebo? That was the concept of the trial and the question we aimed to ask.

We had 3 arms in that trial. We published an interim analysis of that a couple of years ago in which we demonstrated that RBX2660 reduced the recurrence of 

 more than the placebo arm, and that’s highly consistent with the other trials that we’ve run in this space. We’ve run a total now of 5 trials—2 of which are awaiting readout very soon—5 trials in the recurrent CDI [

 infection] space with this investigational product, RBX2660. This trial, PUNCHCD2, showed very consistent response with the other trials.

In addition to that clinical benefit, we also observed a microbiome change from before to after treatment. I’ve introduced the idea that when patients get 

, they have a disrupted microbiome, and what we sought to answer was, have we changed that? Have we provided any restoration toward something more resembling a healthy microbiome? What we saw and published was just that. Among participants who responded to RBX2660, there was a very clear and statistically significant restoration of the microbiome from before to after treatment toward something more resembling a healthy population. We published that last year.

In this study that was presented at ID [Infectious Diseases] Week 2020, we really wanted to take a next step in our translational study and say what effect we have on the metabolome.

We talked a lot about microbiome. The metabolome is all the different functions and metabolites that are being affected by the microbiome. Specifically, in this case we looked at bile acids and the composition of bile acids in the participants’ gut from before to after treatment. Bile acids are interesting to this disease. Typically, our body produces primary bile acids in the liver. Those are converted to secondary bile acids by a healthy microbiome. That’s very important to 

 because the primary acids that the liver produces would normally, if you take those and put them in a test tube, stimulate growth of 

Going back to our garden, they give a growth spurt to 

. But if you have a healthy microbiome, those primary bile acids are quickly converted to secondary bile acids. Again, if we take that and put it in a test tube with 

, we know that those secondary bile acids actually decrease 

. The model that we set out to test is if we have primary bile acids, we would consider that healthier. If we have secondary bile acids, we would consider that less healthy. When we looked at participants from this PUNCHCD2 trial, we found that prior to treatment with RBX2660, they had a preponderance of primary bile acids. That’s not what we consider a healthy balance, but after treatment they rapidly and progressively shifted to predominance of secondary bile acids.

Again, those are the kind of bile acids that are generally associated with repression of 

 growth and proliferation. That was very consistent with the microbiome results we have seen. It was also very consistent with the clinical outcomes that we had seen, meaning we see a restoration of microbiome and bile acid, and concurrent with that, we see a real benefit to the patients in this trial, in terms of the primary end point being met.

This is a very exciting trial. This was the third trial in which we saw this positive benefit for patients. We’ve since run an additional 3 trials, 2 of which are pending readout. We’ve shared with the public the preliminary positive readout from our large phase 3 pivotal trial. This was the first of its type in the area, a large phase 3 randomized, placebo-controlled trial that was aimed at enabling an approvals process with the FDA. The significance of this trial within our developed program was that it’s building more and more evidence that we would eventually take to the FDA and argue for an approval of this investigational product.

For the industry and for the field at large, particularly for the patient population, it’s another demonstration that this type of standardized process and product has the potential to be a game-changer instead of just treating over and over with antibiotics. We’re aiming to have an approvable product that can restore the microbiome and get patients out of that vicious cycle of recurrence.

From a clinical perspective, we also saw that not only in the first 8 weeks did we see a benefit for reducing recurrence in this trial, but if you carried that out to 24 months—it wasn’t a primary end point of our trial, but it was something that we monitored prospectively—24 months after receiving treatment, the primary responders generally remained recurrence free. What we’re seeing is a durable long-term reduction of 

recurrence. That’s very exciting, and the clinical significance for the field is tremendous if we can get approvable products like this in their hands.

As for how we would anticipate this being administered in the clinic, I think that’s relatively straightforward. We anticipate it being administered per whatever label we would be granted. We won’t discuss the labeling today, but that’s what we would aim for, and that would be highly consistent with what we’ve tested in the trials, which is about reducing the recurrence at 8 weeks after treatment among patients who have already received standard-of-care antibiotic therapy.

RBX2660 for Recurrent Clostridioides Difficile

 We live in a society where it’s recognized that antimicrobial resistance is increasing. It’s proliferating, it’s being spread. Any options like a microbiota restoration treatment that would allow us to get away from more and more antibiotic usage does have the potential to reduce the spread of antimicrobial resistance.

We also know that in this patient population that is afflicted with 

], they’ve had an awful lot of antibiotics. Whatever they had originally that required them to have the antibiotic that stimulated the first 

, in addition to the treatments with antibiotics they had for the 

, this breeds antimicrobial resistant bugs, therefore, many of these patients are colonized in their gut with bacteria that carry resistance to various antibiotics. We were interested in looking at that in all our trials.

The trial that we talked about at ID [Infectious Diseases] Week 2020 was a different investigational product. It’s called RBX7455. In addition to RBX2660, this was a formulation we developed to be orally administered.

It’s a very interesting capsule in that it has some characteristics that are amenable to the patient. The number 1 being that it’s not frozen. It doesn’t have to be stored in the freezer. What that means is that participants or patients could come to the clinic, receive their first treatment, take it home, and self-administer outside of a health care setting. That’s exciting to us, and we were excited in this trial to test that. We’ve published the clinical findings from this trial.

This was a trial that was investigator-sponsored. It was a phase 1 trial conducted by Sahil Khanna, [MBBS, MS] at the Mayo Clinic in Rochester, Minnesota. It was an open-label trial designed to test 3 different dose ranges of RBX7455 in patients with recurrent 

All 3 doses had equivalent efficacy, an average of 90% recurrence free at 8 weeks after the last received treatment. That was also highly consistent with our other trials in which we saw a benefit for reducing CDI [

 infection] recurrence. The lowest dose in that trial that we administered was 2 capsules twice a day for 2 days. To me, that sounds much more like a standard drug. The patient experience is aimed at being something very manageable. The second day of those doses, as I mentioned, could be administered by the patient, self-administered outside a health care setting. We’re really excited about that possibility because it takes us to many indications beyond 

Given the clinical outcome of that trial, we had also looked at and published a very recent publication, in press right now in 

. We saw a similar microbiome response to the trials I talked about earlier. Specifically, we saw that patients who came to the trial, baseline is what I like to call them, before they received the treatment, they had a very disrupted microbiome. They had a lack of the good commensals and an overgrowth of bacteria that you wouldn’t want to see in a healthy microbiome. After treatment, that had shifted to become something much closer to a normal healthy microbiome that you would associate with resisting colonization by 

In light of that, we also wanted to look at that data. We look at the microbiome compositions by taking fecal samples from the patient and sequencing them so that we can read the sequence of all the bacteria in their gut. That tells us which bacteria are present. What we can also do is we can dig into those sequence and say among those bacteria present, what types of genes do they carry that would give them resistance to specific antibiotics?

Scientists can go into those genomes and find that there’s a gene that gives it resistance to a high-powered antibiotic, something called an 

, or extended spectrum beta-lactamase. That’s the type of bacteria that you hear about on CNN or in 

 that are pan-resistance problems. We found that before treatment, patients had a lot of these types of genes that confer resistance to antibiotics. After treatment, that had decreased to where participants had far fewer of these genes, and they were more similar in the total amount of genes they carried compared to a healthy population.

We also found that in a few of these patients you could find specific genes, one of those being the 

 gene. We found one of those in a patient prior to treatment with RBX7455, and it was not there after treatment. This is consistent with data that we’ve generated for RBX2660 in our other trials where you really see that there’s a potential for decolonization, of allowing those to be bred out from the microbiome from before to after treatment. That’s very exciting to us because not only are these patients no longer harboring or having resident in their gut a really dangerous pathogen, but it also presents the idea that we can go in to other populations beyond 

 where these types of bacteria could crop up at any time and cause a serious problem. We might be able to go into those patients and decolonize them as our specific aim of a treatment, and we’re exploring that in other trials right now.

Antibiotic Microbial Resistance & RBX7455 for C Diff

 There’s a lot of exciting work going on. The 

] field is very active right now. One of the things that we’re all waiting and watching for is another antibiotic in phase 3 clinical development called ridinilazole. It had a positive phase 2 trial. We’re all eager to see the phase 3 data.

More importantly, among Rebiotix Inc and parent company Ferring Pharmaceuticals and all the other companies running trials in this space, we’ve seen a real explosion of the knowledge around 

 as a pathogen, its pathogenesis, the disease, and how it affects patients. Our knowledge has increased exponentially over the last several years, and that’s really good for the patient, the health care system, and drug developers. We’re all learning more about how to manage this disease and how to improve it.

When we looked at that microbiome data for all of our trials, we were able to find a very clear pattern, which we call a Microbiome Health Index [MHI]. This is a single number based on the compositions of various bacteria, and we use it as a biomarker in our trials. What you see is that patients who come into the trial have been antibiotic treated, so they’re a good example of an antibiotic-induced dysbiosis, or antibiotic damage to the microbiome.

They have a very low MHI. After treatment, their MHI goes up much higher. Then we can actually derive a cutoff where we say, “OK, above this certain MHI level—the same way you would for other markers like blood pressure—is really indicative of a healthier microbiome. But if you get below that, you’re really starting to show some signs of antibiotic destruction or damage to your microbiome, which may trigger other considerations.”

 and ask the question for other populations, can you start to gauge what level of damage a certain antibiotic did to the microbiome? We’re starting to apply the MHI to those types of questions, and we’re seeing that the MHI is quite effective at telling a difference between what are thought to be microbiome-sparing versus microbiome-damaging antibiotics. That’s a small example of how this explosion in all the translational biology and clinical research around 

 has developed knowledge that extends beyond 

 and pushes this forward as a community, both for the patients and the health care providers, as well as the drug developers.

We’re seeing a lot of trials approaching the late stage. For our phase 3, we notified the public that we do have a preliminary positive phase 3 readout. We’re expecting to see positive data next year that we will roll out at a major conference. We will also be starting a large trial with the capsule formulation that I just mentioned. There’s a lot of excitement. When we talk about that phase 3 for RBX2660, that’s pivotal because it represents potentially the first approval in this microbiota-based live biotherapeutic class. That opens the doors for a lot of additional approvals, a lot of additional investments, and potentially transformational approaches to health care.

Paradigm Shift in the Management of Clostridium Difficile Infections

Clostridioides Difficile Pathogenesis & Manifestation

Managing Recurrent Clostridium Difficile Infections

RBX2660 for Recurrent Clostridioides Difficile

Antibiotic Microbial Resistance & RBX7455 for C Diff

Clostridioides Difficile: Exciting Developments