I think for, you know, the last couple years have been tough for everybody and coming back face-to-face and talking about all this new and exciting stuff going on at Arrowhead is really, really great. Thank you for joining us. Safe harbor. The obligatory slide, what we're gonna be talking about today includes forward-looking statements, so please refer to all the risk factors in our SEC filings. Here are our panelists. We've got a great group to talk to you guys today about our pulmonary platform and the two new pulmonary products, MUC5AC and RAGE, and then our newest announced program today against MMP7 for IPF. The external panelists are Dr. Mario Castro and Dr. Matthias Salathe, both from University of Kansas Medical Center.
Both are experts in the field of muco-obstructive and pulmonary and inflammatory pulmonary disease, so we're very lucky to have them today. From the company, myself, Chris Anzalone, our CEO, Erik Bush, our Group Vice President of Biology, James Hamilton, our Senior Vice President of Discovery and Translational Medicine, Javier San Martin, our Chief Medical Officer, and Anjali Sharma, our Senior Director of Commercial. Here's the flow for today. Here's what we're gonna cover. Chris will go into an overview of the company, and kind of where we've come over the last few years. Erik will talk about some non-clinical pharmacology for the new programs that we're bringing into the clinic. James will talk about learnings from the ENaC program and non-clinical toxicology results. This is important, so keep an eye on this.
This is what has enabled us to reemerge with the pulmonary platform very confidently. Doctors Castro and Salathe will talk about mucins as well as the RAGE target in obstructive and inflammatory pulmonary disease. Javier will talk about our general clinical development plan for MUC5AC and for RAGE. We'll start with the initial phase I studies and then go on to how we're thinking about phase II and beyond. Anjali will talk about some of the market research we've done over time about pulmonary generally about route of administration, and about what's really needed. You know, where's the unmet need in these diseases. Chris will do some concluding remarks, and then we'll o pen up to your questions for the whole panel. Hold your questions up until the end. Now I'll turn it over to Chris.
Thank you, Vince. Thank you all for coming today. It's great to see all of you. It's great to be out again. I'm the least important speaker today, and so I will be brief. About four and a half years ago, we had an R&D day here in New York to introduce the TRIM platform. It was a different time in our in the history of this company. The prior year, we had discontinued the DPC platform. Our stock was trading for around $3 a share. I showed the following slides on the Hubble telescope. When Hubble was developed, when it was launched, they decided to see what it could do.
To do that, they looked for some corner in the sky that didn't seem to have anything going on. They found this little area shown here with the red dots, right near the Big Dipper that seemed to be relatively devoid of objects. As they looked closer, they found this little area here that looked truly devoid of objects. As they continued to look closer, sure enough, it was empty. Perfect. Let's aim Hubble there and see what we see. What they saw was not a void, but tons of objects. It's a mind-blowing concept, right, that they found this tiny little corner in the sky that appeared to be empty, and it turns out it was not empty.
The point is, they had developed a tool to see what others could not. Now NASA was able to see what others couldn't. That was an important concept to us and really resonated with us. Four and a half years ago, when we talked about the TRIM platform, what we were really talking about was developing a set of tools that allowed us to see what others could not see. These tools then enabled us to develop the TRIM platform. They enabled us to design, I think, the most potent triggers on the planet. They enabled us to make important new lung-directed drug candidates. It held out the tantalizing possibility that we could bring RNAi outside the liver.
Today's presentations are really an outgrowth of that concept of four and a half years ago. We are expanding RNAi outside the liver. We are going to where diseases are rather than just going to the liver. It's also the presentations today are also an expression of our commitment to continuous innovation. We learned a ton from our first clinical pulmonary program, ARO-ENaC, and we used those lessons to inform what we're doing now in the platform. In fact, you know, in less than a year since we stopped that program, we'll be treating human subjects in these next two programs. Today, I hope you walk away with a few thoughts. First, that the targets that we're addressing are important and potentially powerful.
Second, that there is substantial unmet medical need that we'll be addressing. Third, that our non-clinical data suggests we've got a pretty good shot of success. Fourth, our clinical plan is appropriate and achievable. Fifth, that we're just getting started. This is a broad platform we see. We can see within this platform not just two or three drugs, but eight or nine or 10 drugs. We are looking forward to expanding this platform to bring it to where patients really need it. With that, I will hand it over to Erik Bush.
Thanks, Chris. Do I have a pointer here, or will I just have to gesture?
There's a green one.
Is there?
Yeah. Green one.
Green button. Okay, good. Make sure we have all that. Does this advance? Okay. Good morning, everyone. A brief reintroduction to the TRiM delivery platform for pulmonary delivery of therapeutic sRNA triggers. At its core, the platform is comprised of a therapeutic sRNA, or as we refer to it, an RNA interference trigger. Critical to the success of these triggers is an algorithmic approach that selects the most potent and specific triggers and avoids any off-target. Our approach is unchanged to what we've used in the past. Critically, we're now beginning to employ some additional modification chemistries that improve the duration and potency of our conjugates, and this will be highlighted in our ARO-RAGE program.
Then finally, the third critical element of the pulmonary delivery platform is a targeting ligand to the alpha v beta 6 integrin, a small molecule that facilitates internalization by epithelial cells after inhalation. One brief comment on the inhaled platform and the environment of mucus. We just want to highlight that the physicochemical properties of the TRiM conjugates for inhalation are highly compatible with mucus transit. Critically, they are of a very small size, 3 nm-10 nm in size. Much smaller in order of magnitude or more smaller than the mesh size of mucus, which is 100 nm-200 nm in size. By comparison, respiratory viruses or lipid nanoparticle formulations for mRNA delivery are much larger on the order of 100 nm.
The TRIM conjugates have a negative charge, which is highly favorable in terms of anionic compounds are minimized with respect to electrostatic interactions with mucus, and they're soluble. We have, in addition, multiple lines of evidence showing efficient delivery through the mucus in vitro, through mucus layers, as well as in models of airway mucus hypersecretion, a delivered drug having a robust pharmacodynamic response. With that, I'll proceed to our first program we'll be talking about today, which is targeting the receptor for advanced glycation end products. Dr. Salathe will be giving us a much deeper dive into this target later this morning. I just want to briefly introduce this receptor. This is a pro-inflammatory pattern recognition receptor that's highly abundant in the lung epithelium and expressed at very low levels outside the lung.
It's important in the lung because it has a very large repertoire of pro-inflammatory ligands that it's able to interact with, and we'll be hearing about that. Critically, signaling through this pro-inflammatory receptor culminates in canonical signaling through NF-κB and other pathways that produce cytokines, reactive oxygen species, mucin production, as well as reinforcing expression of the RAGE receptor itself, which amplifies and perpetuates chronic inflammation in the lung, in combination with other canonical signaling pathways. We'll hear more about it. What has made this an exciting target for respiratory inflammatory disease is the knockout phenotype of the mice. These mice offer nearly complete physiological and histological protection from various allergic asthma stimuli.
Traditionally, this target, despite its great interest as an inflammatory target, has been quite challenging to drug with traditional small molecule approaches, and this is largely due to its structure. Its structure is not a canonical G protein-coupled receptor with a discrete binding pocket for a subset of ligands. The structure is an antibody. It's an immunoglobulin structure, which allows many interaction motifs with a wide variety of ligands, thus making it difficult to block with a discrete agent and making it ideal for a therapy like RNA interference. Finally, this target is quite interesting because it's abundantly expressed in the lung, and it also is shed into circulation in a form called soluble RAGE, which can be readily detected in serum from treated animals or patients.
It allows a circulating biomarker of target engagement for delivered lung inhibitors. Let's move directly into rodent pharmacodynamic data with a RAGE silencing trigger conjugate. These rats received a single inhaled dose of a RAGE conjugate at day zero and were at 0.5 mg per kg delivered dose in the lung. Just a single dose, and we're following these animals out for two months post-dose. In blue, we're tracking whole lung expression of the RAGE receptor on mRNA. What you can see is that by day three, post inhalation, greater than 90% of the whole lung mRNA for RAGE is fully depleted, and it remains in that range for at least 60 days after dosing. We'll see more detailed recovery kinetics in a moment. Importantly, serum RAGE also falls. It takes time to delete the protein pool from the lung.
By a month post-dose, serum RAGE also reaches undetectable levels, showing that in the rats, the vast majority of soluble RAGE that can be detected originates in the lung. This is confirmed by immunohistochemistry, looking at RAGE protein expression, in the lung at day 36 post-dose in these rats. You can see that this pro-inflammatory receptor has been completely removed from lungs of animals dosed with this RAGE conjugate. Moving on from there, we were interrogating pro-inflammatory effects through RAGE signaling and seeking to phenocopy what's been observed in the knockout mice. We do this in rats with an allergic asthma model. These are rats that are sensitized to a fungal allergen, Alternaria alternata, and can be delivered to rats to provoke an allergic asthma response, inflammation, and other sequelae.
What we see here is that rats receive this Alternaria challenge. They get a robust inflammatory response. We can do alveolar lavage in those animals, retrieve samples, and count inflammatory cells and measure cytokines. What we observe is that as expected with this fungal allergen challenge, we get a robust eosinophilia. We see neutrophils as expected. In animals that receive that single dose of RAGE silencing conjugate, and we've depleted this inflammatory receptor, we see very robust reductions in eosinophils in lavage and neutrophils and reductions through a wide panel of pro-inflammatory cytokines that can be detected in the lavage such as IL-13, MIP-1α, and IP-10. Again, we have clearly phenocopied the mouse knockout phenotype now in a rat model, but using an inhaled RNAi conjugate.
On the basis of this work, we've moved forward with ARO-RAGE, our clinical candidate that we intend to take forward. This was a study done with a single inhaled dose in cynomolgus monkeys at a deposited dose of 1 mg per kilogram. What this slide simply shows is that after inhalation, we look at multiple regions of the lung, left lung, right lung, proximal, medial, distal, all throughout the lung, we see very robust, greater than 90% silencing throughout the lung. Next slide. Ooh, I guess I have the slide. From there, we moved on to dose response studies in cynomolgus monkeys. These monkeys received a single inhaled dose of ARO-RAGE, ranging from 0.13 to 0.47 mg/kg deposited dose.
We take the animals down a full four weeks post-dose, where again, we expect depletion of the protein. We see indeed here by Western analysis greater than or equal to 90% depletion of lung protein in cynomolgus monkeys at doses of 0.3 deposited dose or higher. From there, you know, given the potency of ARO-RAGE and these RAGE conjugates, we wanted to explore alternative routes. ARO-RAGE is intended for inhalation, but we were curious to. You know, our GalNAc conjugates are so effective, sub-Q for delivery to the liver. We wanted to explore the effect of these conjugates towards the lung. Indeed, we can see efficient delivery to the lung at higher exposures when given sub-Q.
In the lower left-hand panel, these are rats that received a dose response, a single dose of a RAGE-targeted conjugates. What we observe at a week after dosing, roughly 70%-80% whole lung knockdown of RAGE can be achieved via a subcutaneous route of these RAGE-targeted conjugates. Importantly, this knockdown was entirely dependent upon the epithelial targeting ligand. Without the targeting ligand, there was no delivery and knockdown of RAGE in the lung. A little bit higher dose, of course, to achieve knockdown. We see about 70%-80% knockdown at the 15 mg/kg dose level. What we recognized was that with repeat weekly doses, in this case three weekly doses at 15 mg/kg, we could start to build even deeper knockdown over time.
What this slide shows is the pharmacodynamics of silencing and recovery for inhalation and comparing that to what we see with sub-Q. Again, this is rats on the left having a single inhaled dose of our RAGE conjugate here on day one. As I showed you before, now we're monitoring serum sRAGE as a marker of target engagement in the lung. We have full depletion by a month post-dose. It stays near the limit of detection for about two months post-dose. The next two to three months or so, we have slow recovery. This knockdown and recovery kinetic are roughly analogous to many of the GalNAc conjugates for liver targets. Now, by sub-Q routes, again, we have to give a bit higher doses.
In the upper right-hand panel where we're looking at a weekly dose strategy, where we observe that at 15 mg/kg and 10 mg / kg doses, not weekly, every other week, we could slowly build and achieve very deep lung silencing of our RAGE target that was maintained. Lower doses at 5 mg/kg and 2.5 mg / kg were below the threshold to achieve the very deepest knockdown. Those 15 mg/kg and 10 mg per kg dose levels could also achieve deep lung silencing of RAGE given monthly, but it took a longer period of time, perhaps up to three months, to achieve that knockdown via the subcu route. Just demonstrating the possibility of subcutaneous.
One final slide before we leave RAGE, just to highlight the improved pharmacodynamic response of this next generation RAGE conjugate versus our first generation ARO-ENaC targeting conjugate that was designed to silence the epithelial sodium channel. Here we're looking at rat ENaC mRNA expression following doses of ARO-ENaC on days one and two, it's 0.7 mg / kg. We see a nadir of roughly 60%-70% whole lung silencing of ENaC mRNA by 2 weeks, and then a slow recovery over the next couple of weeks. For ARO-RAGE, the differences are quite striking. Again, deeper knockdown at a lower exposure with a slower recovery kinetic.
From there, I'd like to move quickly on to the next program we'll be hearing about today, which is a therapeutic strategy to silence a pathologic mucin MUC5AC for severe asthma. MUC5AC is one of two genes that encode gel-forming mucins that are secreted by secretory epithelial cells in the airway. These two gel-forming mucins together mix and form the mucus layer that rise on top of the airway surface liquid in the lung that is moved by cilia to create efficient mucociliary clearance. Now, what's important about these two isoforms is that they're quite different. MUC5B is the predominant form of mucin expressed in lungs. It comprises about 90% of mucin at baseline. It's constitutively expressed and required for normal lung clearance. In knockout animals, it's lethal.
They do not have efficient mucociliary clearance. The form we're talking about today is MUC5AC. It's a minor component at baseline, comprising about 10% of the mucin protein in the gel. Knockout of this mucin, as we'll hear about, is completely normal, but is highly induced in settings of inflammation like asthma, allergic asthma, COPD. Here you can see this mucin stain in inflammatory asthma models. What that means is that mucin hypersecretion, and particularly MUC5AC upregulation, disproportionately contributes to the pathology of muco-obstructive lung diseases like severe asthma. What we are proposing here is the very first therapeutic approach to directly and specifically silencing expression of this pathologic mucin gene.
There's a great deal that's been written on the role of mucus in asthma, and we're very fortunate to have Dr. Castro here today to tell us more about it. I will not sort of preempt much of his discussion, but the core themes here are that the disproportionate upregulation of this MUC5AC mucin creates a highly sticky mucus that's hard to clear by airways, and leads to mucus plugging and airway occlusion, driving airway dysfunction in these diseases. Touch briefly on the knockout mouse phenotype. Again, Mario will walk us through this data again, but it was a key insight into this specific mucin's contribution to asthma. These knockout mice were found to be completely protected from airway hyperresponsiveness in late phase allergic airway response.
This is what happens with an allergic reaction in the hours or even days after an allergic asthma attack. These knockout mice, instead of having these muco-obstructed airways with high amounts of MUC5AC, there was no airway obstruction. When these mice were challenged with a bronchoconstrictor to further provoke airway dysfunction, they were completely protected. This shows that the combination of MUC5AC plus bronchoconstriction were a key driver of late phase airway dysfunction in the mouse. That's critical because we'll show you a little bit more data here on our program. When we started on this journey for MUC5AC, we started with our own allergic mouse models. These are mice that are sensitized to various allergic asthma allergens such as house dust mite allergen or IL-13.
At baseline, these mice express almost undetectable levels of MUC5AC mRNA. Then when they're subjected to this allergic stimulus, there's a very strong induction of MUC5AC mRNA. If animals have been pretreated in the prior weeks with a trigger silencing MUC5AC expression, we can block, to a very significant extent, much of that induced MUC5AC expression in the range of 70%-95%. This is both in mice as well as cynomolgus monkeys. What really comes through is if you look at the protein level by immunohistochemistry, here we're staining for MUC5AC protein in red. In normal airways, these are the airway epithelial cells secreting the mucins. Clearly, with an allergen, you can see how much of this mucin is being produced and stored in secretory granules and airways.
With the trigger on board, we're silencing much of that, upregulated mucin expression. Where it really matters is in animal models of airway dysfunction. What I'm showing you here is data in a sheep model of allergic asthma with ARO-MUC5AC, our clinical candidate that you'll be hearing more about today. This large animal model, the sheep model, is a standard model for the study of asthma drugs. These sheep are sensitized. They're allergic to a nematode allergen, Ascaris. If these sheep get inhaled Ascaris antigen, they have a classical asthma attack, an allergic asthma attack, which is comprised of bronchoconstriction. They have inflammation and an immune cell recruitment. There's robust secretion of mucus and airway occlusion, airway dysfunction, and these animals respond to standard of care therapy.
They highlight many of the key aspects that are seen with asthma patients. What I am showing you here is the late phase response of these sheep in the four to eight hour period after this allergic asthma attack. First focusing on the black line here, we're observing lung resistance. This is lung mechanics of sheep, conscious sheep, and we're studying how occluded their airways are, how much lung resistance there is during this period. What you're seeing here is a classic late phase response, that after this allergen challenge in the four to eight- hour period, you have an increase in lung resistance as inflammation and mucus come in, and you have this essentially doubling of lung resistance during that period.
What we observed in animals that received ARO-MUC5AC in the weeks prior to this Ascaris challenge is a dose-dependent reduction in a late phase response read out by lung resistance. Critically, we also studied these animals at 24 hours post-challenge. Now, at 24 hours post allergen challenge, lung mechanics have fully returned to baseline. These animals are breathing normally. For all intents and purposes, their lung mechanics seem to be normal. You can superimpose a stress on those animals by giving them an inhaled bronchoconstrictor to tighten up their airways. In untreated animals, we measure this by giving this inhaled bronchoconstrictor, carbachol, and measuring lung resistance again and seeing how many breaths of that bronchoconstrictor those animals need to take to get a five-fold increase in lung resistance.
In normal sheep or in sensitized sheep that have not received that allergen challenge, it takes them about 22 breath units to get that airway resistance. In the 24 hours after this Ascaris challenge, you do the same bronchoconstrictor challenge, it only takes them 12 breaths to get this airway resistance. This is the classical definition of airway hyperresponsiveness. And what we saw at all dose levels with ARO-MUC5AC-treated sheep is that there was no evidence of airway hyperresponsiveness in the 24 hours post-dose period. This is a clear phenocopying of the mouse data that I showed you previously with the MUC5AC knockouts. This is strong evidence that MUC5AC expression is playing a critical role in airway hyperresponsiveness in the sheep model.
We also did additional controls looking at, for instance, a version of MUC5AC where we chemically modified the sequence to prevent its loading into the RISC complex and gene silencing, and that was entirely inactive in this model and looked just like untreateds. Before we leave MUC5AC, I just wanna highlight the fact that beyond asthma, mucus hypersecretion underlies a wide variety of mucobstructive lung diseases such as COPD, non-CF bronchiectasis, cystic fibrosis. When we examine mucin concentrations in all these patient groups, we see a disproportionate increase in MUC5AC, highlighting its critical role in driving the central pathophysiology of all these mucobstructive diseases. Now, moving on quickly to our new program in idiopathic pulmonary fibrosis. This is the first time we've spoken about this program.
This program is designed to silence a secreted endopeptidase, matrix metalloproteinase-7, in idiopathic pulmonary fibrosis patients. Now, matrix metalloproteinase-7 and its relationship to IPF has been well established. It is one of a very large diverse gene family of proteases with many different functions. Critically, it is the most or one of the most upregulated genes in patients that have idiopathic pulmonary fibrosis. It is a validated or perhaps one of the best or the best biomarker for disease severity and progression in idiopathic pulmonary fibrosis. Critically, it's been understood for quite some time that it plays multiple roles in IPF pathogenesis as well. It's a therapeutic target. It promotes inflammation, aberrant epithelial cell repair, fibrosis. We'll see some of these data shortly.
It's been known for some time that knockout mice for MMP-7 are robustly protected from bleomycin injury, which is a model of IPF preclinically. Again, why aren't there drugs to MMP-7? Well, largely because it's hard to make isoform-specific MMP-7 inhibitors. This class of enzymes are highly related in their structures of the catalytic domain, and specific inhibitors to MMP-7 alone simply don't exist. Moving forward into some of the pharmacology with silencing MMP-7, we use a rat model of idiopathic pulmonary fibrosis. It's a standard model of bleomycin injury, which causes inflammation and fibrosis. In this model, we give a single inhaled dose here at 1.4 mg / kg of a MMP-7 silencing trigger.
We wait two weeks and then give a bleomycin injury, in this case, two doses of bleomycin separated by a week. After this bleomycin injury, there's a phase of robust inflammation that transitions to fibrosis. In IPF patients, this persists, of course, but in the bleomycin model it spontaneously begins to resolve, and we'll see that in the data. What's critical here is that, with bleomycin injury, there's an induction of MMP-7 expression here at a week after the last bleomycin dose on day 28. By day 42, it's beginning to resolve a little bit in this dynamic model. What's important is that, dosing of the MMP-7 trigger on day one is leading to significant suppression of that induced MMP-7 expression at both day 28 and day 42, shown here.
What does this mean functionally for fibrosis and function? The most important element to study in the preclinical models of IPF is evidence, histopathological evidence of fibrosis. This is done by assigning what's called the Ashcroft Pulmonary Fibrosis Score. I'm showing you here images of lung parenchyma from control animals bleomycin injured with these fibrotic lesions, and then as apparent having the MMP-7 trigger silencing in these animals, there's significantly less fibrosis. This was sent out to an external CRO that scores these slides blindly for us. You can see here this Ashcroft score assigned from 0 to 5. With bleomycin injury and no treatment, most of the animals score as severe fibrosis. With MMP-7s silencing on board, most of the animals here at day 28 are in the moderate to mild category.
Furthermore, one of the key pathologic drivers of MMP-7 in IPF is thought to be through promotion of inflammation. Critically, what we observe in this model of bleomycin injury, robust inflammation, and this can be read out by doing bronchoalveolar lavage and inflammatory cell counts. Like with our asthma models, we see a very profound neutrophilia and eosinophilia in these samples. With the MMP-7 silencing, trigger very significant reductions in both neutrophils and eosinophils, showing an anti-inflammatory effect with MMP-7 silencing. I will have no time to show you the additional data here, but this was correlated with robust protection of lung function as well as increased animal survival in this model. On the basis of this work, we have identified a potential clinical candidate, ARO-MMP7, for idiopathic pulmonary fibrosis.
We've moved this into studies in cynomolgus monkeys. We're showing you here a terminal study with a single, inhaled dose of ARO-MMP7 at four different dose levels. Here at weeks post-dose, we observe again silencing of whole lung MMP-7 in cynomolgus monkeys at doses at 0.6 mg / kg or higher for single dose. We have confirmed MMP-7 protein reductions in BAL samples from these cynomolgus monkeys, which we intend to employ as a target engagement marker in future clinical studies. Finally, we've begun to employ a new model in-house of cultured human precision-cut lung slices, which allow us to take explanted human tissue, incubate them with our TRiM conjugates, and measure target engagement and gene knockdown.
In these human lung slices, we see robust silencing of MMP-7 with our trigger conjugates, and we have established that this is conclusively RISC-mediated and internalization receptor dependent because using a negative control ligand, we see very little evidence of knockdown. If we modify that trigger to block RISC loading, there's no gene silencing. A much larger story around MMP-7 will be presented at the upcoming European Respiratory Society meeting in September. Before we leave the nonclinical section, I just wanna touch very briefly on respiratory virus discovery efforts at Arrowhead. Our HBV programs have taught us that the TRiM platform offers a very powerful approach to directly silence viral gene expression, opening the door to the possibility of programmable antiviral therapeutics for both existing viral disease and emergent diseases.
We are currently working on a candidate for SARS-CoV-2, ARO-COV. Lead optimization continues for that candidate, and it is promising. Behind ARO-COV, we are rapidly developing a pipeline of additional therapeutics for other respiratory viruses that we'll provide updates in the future. My last slide on preclinical pharmacology, I just wanna highlight three key issues here. ARO-MUC5AC and ARO-RAGE are candidates for muco-obstructive and inflammatory lung diseases. Our new platform designs offer improved potency, duration, as well as potential for subcutaneous delivery. We're expanding our therapeutic area opportunities in respiratory into pulmonary fibrosis with ARO-MMP7 and respiratory virus with ARO-COV. With that, I will turn it over to James Hamilton. Thank you.
Thank you, Erik. I'll cover our learnings from the ENaC clinical trial and the non-clinical tox studies, and we'll discuss why we think what we've learned from ARO-ENaC will enhance our probability of success as we progress these new programs, ARO-RAGE, MUC5AC, and ARO-MMP7, into chronic tox and forward. First and foremost, I'd like to state that in the ARO-ENaC clinical study, there was no safety signal identified. Just as a reminder, this study was a phase I/IIa clinical trial. We enrolled 24 healthy volunteer subjects who received drug on days one to three, so a three-day cycle. Then we additionally enrolled four CF patients who received drug on day one to three, and then again on days 22, 23, and 24. They received two, three-day cycles.
Importantly, ARO-ENaC showed no evidence of adverse effects on lung function as measured by spirometry, no adverse changes in oxygen saturation or chest X-rays, and no predominance of AEs or SAEs in the treatment arm. Adverse local lung effects were seen in a chronic six-month rat and a nine-month NHP GLP toxicology package. To review how we dose these animals, the monkeys received drug on days one to three every two weeks, so similar to how drug was administered in the clinic but more frequently. The rats received drug day one to three every two weeks in three of the dose groups. There was a low, mid, and high dose group that received drug on day one to three. There was a fourth group that received drug only on day one every two weeks, and this was actually our lowest exposure group.
At this level, we were able to achieve a NOAEL or a no adverse effect level. However, due to the histopathologic changes noted in the chronic rat study, the ARO-ENaC clinical trial was placed on a voluntary clinical hold. As you might expect, we've spent quite a bit of time thinking about and investigating the underlying mechanism of the tox findings in the rat and the monkey studies, and it appears that the underlying culprit is a process described in the literature as lung macrophage overload. This is a condition essentially of impaired clearance, where the macrophage-mediated clearance mechanism of inhaled particles in the lung following prolonged exposure to otherwise non-toxic particles overloads the macrophages, and it induces recruitment of other inflammatory cells that can eventually lead to lung parenchymal injury.
Importantly, these inflammatory effects are due to a general, particle response. This is not something that's specific to a sequence or even specific to sRNA or oligos. This is found with other, drug classes with particles used in dry powder inhalers and other inhaled particulates. Importantly, there are thresholds that are described in the literature for where adverse effects are seen. The histologic findings from the chronic rat and monkey studies are consistent with the literature description of macrophage overload. Also importantly, our lung histopathologist, one of the leading KOLs in the field, reviewed these slides and took a look and said that these are consistent with classic macrophage overload. Here on the left in the green panel, I describe the classic literature findings associated with macrophage overload, and then in the right two panels describe the findings from both the rat and the monkey studies.
We did see enhanced transfer of particles to lymph nodes in both species. We also saw increased lung weight in both species. We saw evidence of alveolar macrophage accumulation in both species, which in itself is not adverse. We did not see neutrophilic cellular infiltrates, which would have been considered adverse. We saw evidence of alveolar epithelial hyperplasia, but no metaplasia. If you really push the dose at the highest dose levels in some of the rats, there was evidence of fibrosis, but not in the monkeys. Interestingly, this was not called adverse because it occurred at a rate that was only slightly different from the background rate in the animals. Importantly, we did not see any evidence of pre-neoplastic lesions in either species. Here are the thresholds I was describing, and this is from the literature. This plot shows lung tissue concentrations of inhaled particles.
This could be inhaled drug. Based on the literature, the upper threshold of 1 mg/g of lung tissue, this threshold is associated with adverse findings. If you push the dose or the dose frequency and your lung tissue concentrations are above this, you're likely to see adverse effects on histopathology. If you can stay below or close to the 0.1 mg/g of lung tissue concentration, this is the region where the histopath is typically not adverse. Things like increased alveolar macrophages, just evidence of normal clearance mechanisms. In between, as you'd expect, this is a variable region where if you push the dose and get closer to the higher threshold, you're more likely to see adverse findings. If you can stay closer to the 0.1 mg /g threshold, less likely to see adversity. What did we see with ENaC?
These are lung tissue concentrations from the rat six-month chronic tox study. Again, we dosed in four different groups. The first three groups across the bottom are the day one, two, three cohorts, both the low, mid, and the high dose groups. Then on the far side is the mid dose, same dose level administered only every two weeks. No day one, two, three cycle, just day one every two weeks. There was no NOAEL seen in any of the day one, two, three cycle groups, but we were able to achieve a NOAEL when we dropped the dose frequency from day one, two, three, all the way down to day one every two weeks. The take-home message here is essentially less frequent dose administration is critical for avoiding adverse effects and keeping those lung tissue concentrations low.
This can be achieved by avoiding the daily dosing and spacing out the doses. As Erik showed with the data from MUC5AC and from RAGE, we think we can space the dosing out even wider to either every 28 days or even every three months, which should further allow us to avoid adverse findings on histopath. This slide shows the cumulative dose levels over the course of a six-month rat study. Again, the blue dots represent this data from ENaC here on the left, the six-month ENaC rat study. The blue dots show the cumulative dose level where there was no adverse effect. Then anything at or above this green line that represents the threshold for adverse findings on histopath, and this is at about 100 mg/kg , any cumulative dose levels at or above this in a six-month rat study demonstrated adverse effects.
If you extrapolate that to what we are expecting to administer in the MUC5AC and the RAGE chronic tox studies, even at the highest expected exposure, the cumulative dose in a six-month rat study stays well below this threshold for adversity. Here are the top-line results from the ARO-MUC5AC and ARO-RAGE phase I enabling tox study. Importantly, the take-home here is that no adverse clinical or histopathologic findings were seen at any dose level, and the top dose was the NOAEL in both studies. On this slide, this is more of a pharmacology study. This was not a tox study, but the intent of this study was to show that the knockdown of RAGE for prolonged period of time could be well tolerated in the rat.
This is with a rat surrogate trigger administered via inhalation, 0.5 mg/kg , so a pharmacologically relevant dose, not a tox dose. The first thing of note is that we are achieving great knockdown with great duration administering drug every 70 days, and this goes out through about one year. We're able to maintain knockdown at better than 90% in sRAGE, so serum measurement of sRAGE. On the right, you can see we did a takedown at six months, day 170. We're getting great knockdown in mRNA, tissue-level mRNA. Importantly, over the course of this study, which is still ongoing, the treatment was well tolerated in these animals. There were no significant adverse changes in labs, including hematology or chemistry.
Importantly, there was no significant adverse changes on histopath at the day 170 takedown and no evidence of macrophage overload. In summary, we believe the ARO-ENaC experience has helped us to de-risk the future chronic toxicology studies for programs such as ARO-RAGE, MMP7, and MUC5AC. We believe this because we have new clarity around the mechanism of toxicity and how that mechanism correlates with the exposure levels, with a better understanding of the exposure levels where we are likely to see adverse findings. We also believe that we have better triggers. Both ARO-RAGE and ARO-MUC5AC acute tox studies compared very favorably with both GLP and non-GLP acute tox work we've done in other programs.
We think the better depth of knockdown and the duration of knockdown that we can achieve with these newer triggers will allow for overall lower exposure, and this can facilitate spacing out the doses in tox studies but also in the clinic. Then lastly, but very importantly, we have available biomarkers for all three of these programs that can be measured in the sputum or in BAL fluid or even in the blood. This will allow us to better understand dose response and set dose levels and dose intervals in the clinic, but also in chronic tox studies. This was not something that was available with ARO-ENaC. Next, I'd like to hand over the discussion to Dr. Mario Castro. He'll be describing mucins in obstructive lung disease.
Thanks, James. So now we move on to the clinical part of the discussion this morning, and I will be talking about the role of mucins in obstructive lung disease. Really not talking about any of the drug issues, just really setting the stage for the opportunity that is available here in our patients. Just to give you a little bit of background, I'm from Kansas City at the University of Kansas there. This is a shot from our plaza, which is in the middle of Kansas City. We're known for our fountains. We have more fountains than any other city in the U.S. and second in the world only to Rome. These are my disclosures.
In regards to asthma, it's one of the most common chronic diseases that we encounter, and over 25 million Americans are impacted by this. This results in substantial healthcare utilization in terms of exacerbations that these patients are experiencing, leading to emergency room visits and hospitalizations. Unfortunately, even though this is a completely preventable disease, there still remain over 3,000 deaths per year. What is the unmet need in asthma? Despite our work in this field and really come up with great therapies for our patients, we still are not achieving asthma control in about 50%-60% of patients when we've looked at this in longitudinal epidemiologic studies.
This results in substantial number of hospitalizations as noted, significant morbidity and mortality, and a high economic burden in estimated over $25 billion or $25 million per year. There's a subset of these patients that really drive the healthcare cost, and this represents somewhere up to 10% of our patients with asthma that fail to respond to the conventional therapy. These are patients that are using the standard of care therapy, but are really not responding to that therapy. I'll subsequently share some light into why we think they are not responding to that standard care therapy. First, I'd like to talk about where we've evolved in terms of our understanding in regards to how do we approach asthma. It's such a common chronic disease.
It can be misdiagnosed in a substantial proportion of patients, and therefore, we need better tools to identify these patients and segregate them into various categories. We have grouped patients based on clinical characteristics. What we need to know is, you know, not one drug or one therapeutic approach is gonna fit all of our patients with asthma. Therefore, where we've evolved to is really understanding what are these clinical characteristics that segregate the various patients of asthma. I wrote an editorial, we called this the many buckets of asthma because it's not one disease. Because of this phenotypic clustering that we've done in cluster analysis and unsupervised learning analysis in cohorts, we've come up with understanding then perhaps what is the underlying pathobiology that's driving those clinical characteristics.
Therefore, we could then go to a much more precise kind of precision medicine approach in regards to asthma. When we look at these many buckets of asthma, we have grouped them into, two large categories, T2 high asthma, and then the other bucket is basically everything that is non-T2, disease. What do we mean by, T2 and non-T2 is, for those that are not familiar with the immunology, it's based on that, the immune system, the T helper system drives a lot of the, production of cytokines in response to allergens and other stimulants in the airway. We now understand that it's not just T helper cells, but also other lymphocytes that are important in this immunologic response. We have broadened that category to T2 high asthma.
We have made a lot of progress in the left-hand side of this, in terms of early onset disease, and as demonstrated here, in childhood onset asthma, tends to be highly allergic type of patients, and then they develop, eosinophilic asthma in this particular subcategory. However, the challenge right now is to the right-hand of this. We have a substantial proportion of patients that have T2 low asthma that we really don't have good therapy for these various groups of patients demonstrated here. These patients are taking the standard of care, and the medicines are not working for them. How can we tease this out and dig a little bit deeper into the pathobiology of this? Certainly, our understanding for this has evolved over the last couple decades.
Classically, in the past, we just focused on the role of allergens in regards to asthma. Now we understand there are certainly a number of other stimuli to the airway epithelium, including pollutants and microbes that stimulate the immune response. In the classic allergen-driven model, you can see these allergens encounter antigen-presenting cells such as dendritic cells, trigger downstream production of immunoglobulins that are important in terms of IgE production and activation of mast cells and other cells. This has been targeted with an agent called omalizumab, a biologic agent that has now been in our armamentarium for the last almost 20 years. We have a lot of experience in that regard.
Now, when we look at, the other side of this, these T2 cytokines in high T2 asthma, they can produce IL-4, IL-5, and IL-13. These are what we call the T2 cytokines. These are the signature that we use to identify T2 asthma. IL-5 is one of the, important T2 cytokines because it attracts, eosinophils into the airway, and it lets them survive longer and unfortunately do the damage they do to the airway. Fortunately, now we have three drugs that block, eosinophil infiltration into the airways by blocking IL-5. Benralizumab, Reslizumab, and Dupilumab here is a blocker IL-4 and IL-13. The, other one that's not listed here is mepolizumab, which is an IL-5 blocker. this Dupilumab is one of the more recent entries into the market. It blocks both IL-4 and IL-13 by blocking the IL-4 alpha receptor.
The latest entry into the biologic world for asthma is tezepelumab. Tezepelumab has a unique mechanism by blocking upstream this TSLP, which is one of what we call the alarmins. You can imagine when you're inhaling either an allergen or some other insult to your airway, the airway epithelium is the kind of first signal to the body immunologically and releases these alarmins, IL-25, IL-33, and TSLP. What you can see here is in terms of targetable things, there are a number of other non-allergic, non-eosinophilic pathobiology that we haven't really tackled yet in regards to asthma. We believe that this represents 40%-50% of patients that really don't have any of the type two biomarkers that we use in the management of our patients with asthma.
We typically do a blood count and look at the differential. We look for elevated eosinophils in the blood. We do IgE levels, and we look at exhaled nitric oxide. So those are the three biomarkers that we have available in the clinic to identify this type 2 asthma. However, a lot of my patients that I see on a regular basis really don't have these markers of type 2 inflammation. These are, you know, what we currently have in the clinic. Tezepelumab might work in the T2 low asthma because of that proximal upstream effect it has in the airway epithelium. Recent data just released this last year suggests that there's about a 30%-40% reduction in exacerbations. Macrolide antibiotics and bronchial thermoplasty have also been used in a relatively small subset of patients with mixed effects.
The question is here, could we develop new therapies that would tackle this subset of patients with asthma? Now, the other obstructive lung disease which represents a fair amount of my practice is COPD, or chronic obstructive pulmonary disease. It is a major public health problem and a leading cause of disability. It represents over 20 million Americans in the U.S. have COPD that is identified. There is a prevalence around 6%, and it's the third leading cause of death, and now surpassed stroke as a cause of death. Second leading cause of disability, and third leading cause of death worldwide. This does represent a substantial impact in terms of morbidity and mortality worldwide.
Another thing I often say to my medical students is that, you know, all of our COPD patients aren't the typical older elder patient. In fact, the majority of our patients, as you can see here, 70% are less than age 65. Now, certainly when we look at therapies for COPD, we often follow what's called the GOLD guidelines or Global Obstructive Lung Disease guidelines. These are international guidelines we have for the treatment of COPD. We categorize our patients into these four different categories A, B, C, and D, based on their exacerbations and based on their symptomatology. As you can tell from this, the agents that we have are all mostly inhaled therapies, bronchodilator, a long-acting muscarinic agent or long-acting bronchodilator, or a combination of these, and sometimes we throw in inhaled steroid. These are really very limited.
They have very limited impact on airway inflammation in these patients and really are just helping us symptomatically and do not reverse the progress of the disease progression that occurs in these patients. Certainly, there's opportunity for improvement in terms of treatment for both of these diseases, asthma and COPD. We believe that mucus-directed therapies represent really an opportunity to advance our therapies for the treatment of these diseases. We know that biologics do not resolve the disease process. When I stop the biologic, in about 12 weeks, those eosinophils come back, and the disease recurs in these patients. We, as I mentioned, really have ineffective approaches to COPD. One of our thoughts is one of these targets in that non-T2 population is really addressing the role of mucus hypersecretion.
We've known this for a century now, all the way dating back to this quote by Huber in 1922, looking at the outstanding feature of obstructive lung disease is the failure of clearance of these bronchial secretions. These are postmortem cast of the airways, and these are just basically bronchial cast from these airways. What we've learned in terms of comparing a normal individual that dies compared to a fatal is that the airway basically becomes pruned. You can think about the airways as a tree, and you can think about the branches in that tree allow air circulation and ventilation and a gas exchange. You can see in asthma patients, there's basically a pruning of those branches that occur.
We need better ways to restore this process, and we believe most of this truncation is being due to the mucus that's obstructing those airways. As nicely outlined a little bit earlier, when we look at those mucins in the airways, there really are only a few targets. There are a number of mucins that are important in terms of the periciliary layer here. In this mucus layer, there's really two targets here, MUC5AC and MUC5B. As what's demonstrated here in terms of disease progression is that we believe there's this normal kind of homeostasis of the ENaC channel and other channels here that maintain the hydration that is important in the airway. You have this normal mucus layer here and cilia beating along to progress that mucus normally.
Well, in the muco-obstructive lung disease, what happens is that often there's a failure of this hydration that occurs. There is a thickening of the mucin layer that can occur because of what's referred to as goblet cell hyperplasia. Therefore, the mucociliary clearance is impaired markedly in these patients, leading to the symptoms that our patients experience in terms of cough, airflow obstruction, leading to increased risk of infection, in these airways. We believe that the mucus really that is predominantly in these airways, MUC5AC, is one of the key players in terms of the causation of muco-obstructive lung disease. Let's delve a little bit deeper into MUC5AC.
What's demonstrated here in COPD patients, on the far left is that if you have increasing severities of COPD, you have increased expression of MUC5AC in the sputum that you can detect in these patients. There's really no difference here in terms of MUC5B. So as we'll talk about, we believe that this is in essence, kind of a mucoprotective mucin, whereas MUC5AC is an inducible mucin protein in these patients. Now when we look at patients with asthma, again, very common thing we'll do is we'll look at the ratio of MUC5AC to MUC5B.
This actually is inversed here, so they're looking at MUC5B to MUC5AC, and you can see in asthma and in the setting of an exacerbation, it's significantly reduced. Now look at cystic fibrosis, another mucobstructive lung disease that Dr. Salathe will be talking about subsequently. You can see here in terms of patients with cystic fibrosis, again have an elevation of that MUC5AC, and in the setting of an exacerbation, again, elevation of the MUC5AC.
Lastly, another opportunity for muco-obstructive disease are patients that have bronchiectasis. Bronchiectasis is basically dilatation of the airways that's due to recurrent pneumonias. In the past, we used to see this a lot from tuberculosis, but nowadays we still see this in patients that have recurrent pneumonias, and it's not related to cystic fibrosis, but again, showing this elevation in MUC5AC. Again, this ratio, we believe is very common across this muco-obstructive lung disease and is showing this increased production of MUC5AC in relationship to MUC5B. Well, what about genetic predisposition?
There are now been several GWAS studies or, genome- wide studies that have looked at, various genetic causes, potentially of asthma. We have not found one thing. It is, you know, likely a very complex, multigenic, causation of this disease. In this GWAS study, which was, recently published in Lancet, it showed in a large population, this was in Europe, 5,000 cases, 25,000 controls, that there were three different, targets that met their, criteria for significance, which was set at a p-value of - 10 to the 8th. One of them, these three was MUC5AC, suggesting that there are a subset of patients that have a genetic predilection to development of, muco-obstructive lung diseases like asthma.
When we think about this mucobstructive lung disease, one of the drivers behind that is changes that we're seeing in the airway epithelium in the airways of our patients, in this case, a fatal case. One of these cells that we have identified is the goblet cell. This is turned on by IL-13, one of those T2 cytokines that we were talking about. When that goblet cell is turned on, it basically causes increased mucus production and a plug in the middle of that airway. Now fortunately, I don't have a lot of these cases, fatal cases, but I can use a tool that every clinician has in a hospital here in the U.S., which is a CAT scanner. A CT of the chest allows me to identify a mucus plug.
This is in the right lower lobe, in this patient we enrolled in the Severe Asthma Research Program, that I'm part of, sponsored by the NIH. You can see this plug here in this distal airway. Now look at this patient three years later, again, persistence of this plug in that same airway. As we'll show you next, we believe that this is very important in terms of the disease, causation and progression. This is work by Eleanor Dunican, that we published in JCI a few years ago, again, showing, these mucus plugs in the airways. The way we did this, we selected five chest radiologists around the country, to identify these mucus plugs, and they went through every CAT scan.
We had over 200 of them enrolled in our program, and we had them score. If a segment of the lung had a plug, that was a score of one. Well, there are 20 segments in the lung, 10 on the right, 10 on the left. You can imagine this is pretty tedious work in order to do that. As all things go, you know, now there's a software system that does this automatically for you. One of the key findings from this publication was plugs matter in that if you had a high mucus plug score, four, just four out of those 20, that correlated with a marked reduction in lung function. This reduction in lung function, this mean change in lung function, is considered severe airflow obstruction by a clinician.
You can see that we believe that, again, in this cross-sectional analysis, that there is a potential role for the causation of airflow obstruction in our patients with asthma. Recently, this was just published, this month, by Monica Tang, looking at this now over time. We've been fortunate to follow this severe asthma cohort over a three-year period of time, and you can see the mucus plug score really hasn't changed over a three-year period of time. This is what we call a Sankey plot, where you look at these patients and how they change over time. About a third of patients had these high mucus plug scores. Again, trying to get to that precision medicine approach of what are the subset that we can really target?
You can see majority of these patients, a third still out here three years later. There's a little bit of a change back and forth here, but the majority of patients are staying in that category. What we demonstrated here in Monica's paper was that this does matter because when you look at the change in lung function here by three different parameters, and you look at the change in MUC5AC score, there is a direct correlation between an increase in the MUC5AC score and a decrease in lung function, an inverse correlation. Suggesting again causation in terms of progressive loss of lung function in our patients and some other more advanced CT metrics that we're using in quantitative CT, we are also demonstrating relationships to air trapping, which makes a lot of sense, as well in regards to mucus scores.
Well, what about COPD? Is the same thing true in COPD or not? Again, work led by Eleanor Dunican in our group in the Severe Asthma Research Program. Again, in COPD, you can see here, the mucus plug score again from 0 to 20. A pretty interesting group here in terms of these scores in comparison to the healthy individuals here. Then there's another group here which I think is very interesting and potential target is smokers that have not developed airflow obstruction. Just in recent years, we've identified that these patients are actually quite symptomatic. They have frequent cough, shortness of breath, and chest tightness, and these patients really are not abnormal in terms of lung function testing, yet have significant mucus plugging.
Again, that same Sankey plot that you can see in this particular study coming from SPIROMICS, showing just a year later that there's persistence of these mucus plugs in about a third of patients out to one year. This appears to matter in terms of the mucus score being the highest in the most severe patients by that Global Initiative for Chronic Obstructive Lung Disease categorization of four. They're mostly GOLD 4 type patients. We also use this data to segregate our patients. As I mentioned for asthma, there's many buckets of asthma. Well, in COPD, there's many buckets of COPD as well. You can imagine the spectrum from chronic bronchitis patients all the way to emphysema type of patients.
What the MICA score allows us is to really select that subgroup of patients where mucus is driving that disease process. There is another important role about mucin proteins in the airways, and that's how what role it has in regards to host defense. The most common cause of an exacerbation in both asthma and COPD is a respiratory tract infection, a cold, and most often these are driven by viruses. Now, when you look at COPD patients, you can see at baseline and in the setting of an exacerbation, there's a marked upregulation of MUC5AC in the sputum. And this was just a natural observation study where they took 40 patients with COPD, and a subset of them, they were able to identify a virus and then followed them over time.
Again, we have this biomarker that appears in the sputum in the setting of an exacerbation. In the middle here is a knockout mice of MUC5AC, and you can see here in comparison to the wild type, there is decrease in neutrophilia in these patients, a decrease in inflammatory cytokines, IL-1 beta and IL-6 correlating to the knockout. When you use UV radiation treatment of the rhinovirus, which is the most common cause of the common cold, it basically abrogates that inflammatory response. This indicates to us that there is a role for mucin in terms of attenuating that inflammatory response that we see in the setting of a cold. Again, the most common cause of an exacerbation.
In the far right, you can see here is that when you give MUC5AC, as demonstrated here in the setting of rhinovirus infection in the blue bars here, you see a marked upregulation of inflammatory cytokines, both IL-1β and IL-6. Clearly, mucin is MUC5AC here is playing a role in this inflammatory response in the setting of an exacerbation. Like all academicians, we like a model, and so this model helps me understand, where does MUC5AC play a role in mucobstructive lung disease? I do believe that there's this chronic basal secretion because MUC5AC is a common constituent with protein in the airway.
We know that in a chronic model, a chronic disease model, that increase in MUC5AC now appears to be correlated with chronic airflow obstruction demonstrated in those CT scans that I was showing you by increase in mucus plugs and leading to symptoms in my patients. We know that there's another side of this that you can stimulate increased MUC5AC production in the setting of a viral infection leading to exacerbations of the disease. Airway inflammation basically accelerates this process in our patients, leading to their progressive loss of lung function and progressive disability in that regard. How do I think about this in terms of looking forward, in terms of targets, that are targetable, you know, drug targets in regards to the disease?
Well, I think we can utilize a tool that we have in the clinic in every location, which is the CT scan of the chest, in order to identify the subset of patients that have a high burden of mucus, okay? I refer to this as the mucus-high phenotype. Another way we can use this is looking at, mucus on this axis here versus inflammation. We know that type two inflammation, we have a number of targets in asthma, at least. We have six biologics that I mentioned. But in COPD, we have no biologic that is currently approved, for the treatment of COPD. We have a number of drugs that potentially we can use in this type two high inflammation. We don't have a lot of information about these biologics and what impact it has on mucus obstruction.
It's certainly this category here where you have high mucus score but low T2 inflammation. We have no candidate drug currently in this place. This is certainly a key opportunity for advancement of drugs in this population. Certainly even in this group there may be a role for co-treatment of both T2 inflammation and mucus plug related inflammation. Just to end, I think, as we summarized in this data nicely before, in terms of the knockout mouse, certainly this preclinical data suggestive. Now genetic information in the GWAS data suggesting a causal role for MUC5AC. This clinical tool that we have in terms of measuring plugs that every clinician could use, and there are software now developed to give this information to us based on the CT.
The role that MUC5AC has in a very common cause of exacerbations, which is viral infections. Therefore, I believe that this is a key opportunity to really interrupt this pathway that has quite a bit of potential. As demonstrated, we're certainly focused on asthma COPD, but there are a number of other diseases like cystic fibrosis, non-CF bronchiectasis, and primary ciliary dyskinesia that would represent an opportunity. Thank you. I'd like to end there and turn it over to my colleague. I'd like to introduce Dr. Matthias Salathe, who is the Chairman of Medicine and Interim Vice Chancellor of Research at the University of Kansas. Actually, he's my boss, so I gotta turn it over to him. Thank you.
Well, thank you, Mario. As the bosses always do, right, he set everything up, so I have to do very little at this point in time. I wanted to talk a little bit more about RAGE and how we really believe this could be a great target for asthma. Now, Mario told you a lot about the pathophysiology of asthma and where we have potential, interesting drugs and where we don't. There is a huge number of people, obviously, that still have no treatment in that space. I'm ending up that I don't believe this is the end. You know, asthma is not the only one. Like mucus obstructive disease, RAGE plays a role in those as well. Here are the disclosures, and we're jumping right into this very complicated receptor that Erik already, introduced very well.
It's an immunoglobulin in the superfamily of immunoglobulins, and very difficult to target in terms of inhibitors. You know that there are some developments, but none have really been successful, at least on a clinical basis. Even in animals it's not a great target to go for. There are lots of agonists for that receptor as well. It's not an easy receptor that's just like stimulated by one target. It's targeted by a lot of agonists that then create a signaling pathway in the cell that is complicated, and depending on the cell, can really induce a lot of inflammation in all kinds of pathways. NF-κB was mentioned, but there are lots of other pathways.
The important piece here is that RAGE is really upstream of a lot of the other inflammatory targets. The idea is if you can really take RAGE out, you will be able to suppress inflammation in other areas dramatically. Here's just another example of RAGE, how complicated this signaling can be. You can imagine that really if we can block up here, we don't have to deal with all the complication that comes below RAGE activation. How is RAGE activated? You've seen all the alarmins, the DAMPs and PAMPs, and whatever these agonists are called. Originally it was high sugar that was the classic trigger for RAGE activation. The sugar molecules that created these advanced glycosylation end products that activated the receptor, that was the discovery.
We know now that pollution, cigarette smoke, and allergens, the classic house dust mite and cockroaches and other allergens are stimulating RAGE as well. Here are the classic mucus obstructive diseases again, CF, COPD, and asthma, that we have all these cytokines following, the RAGE activation. Again, if we could take RAGE out, we can decrease inflammation in a lot of these different diseases. As you have already seen, this is a sort of a normal airway with the lung around it, and here is an asthmatic airway. You're not needing to die from the mucus plug, right? You have a lot of mucus. The idea here is not only having mucus, it's this whole inflammatory cascade around the airway that will stimulate the mucus.
It's not only that you target mucus here, it is potentially able to decrease the inflammation that will lead to the mucus obstruction as well. Mario walked into this or through this a lot. We're always thinking about T2 high asthma, and T2 high asthma is really the one we think we can reasonably treat. That's not true either. Not everybody in T2 high asthma is actually treatable, and we have a significant number of patients that are not. As he mentioned, the low T, low asthma, we don't really have therapies for them, and that's a huge issue. That's a large number of people in COPD, obviously, too. This is just another depiction. Here is the T2 high. We understand these pathways. We have created. I take a royal we here. I have nothing to do with that.
Here are people that created these antagonists for these pathways. We really don't have anything meaningful in T2 low asthma. Now, how is RAGE playing a role here? What you're seeing here are biopsies, and these are not quantified, but these are representative biopsies from non-asthmatics, mild asthmatics, moderate asthmatics, and severe asthmatics, and these are stained for RAGE. What you can see easily is that in the more severe asthma you have, the more RAGE expression is there. Now, expression itself obviously doesn't mean it's signaling, so that's always a tricky piece. Here is an experiment in a mouse lung. You see here toluene diisocyanate as an allergen, and it increases RAGE. Then this not great inhibitor of RAGE, FPS, which cannot be clinically used, is slightly decreasing the RAGE expression.
More importantly, if you look here at PAS staining, there is clear mucus production in the allergic mouse model, but you can block it, this production of mucus with this RAGE inhibitor. If you look at these pieces here, you can say the inflammation is reduced and thereby the consequences of the inflammation through RAGE inhibition is actually beneficial. When you look at these alarmins and other antagonists of RAGE, you see they are increased in mild to moderate asthma, it's going up, and severe asthma, it's going up. This is HMGB1. This is a classic agonist of RAGE, but it's also true that that HMGB1 is increased in COPD and in cystic fibrosis, and even worse in cystic fibrosis-related diabetes. This is a agonist that is not unique to asthma, but it's increased. Is S100A9.
This is one of the other agonists of RAGE, increased in asthma compared to healthy controls. You see here also in neutrophilic, meaning T2 low asthma, it is also increased. At least again suggesting that if we can block RAGE, we may be beneficially influencing T2 low asthma. You see here sputum neutrophils, again a correlation between this agonist of RAGE, S100A9. There are lots of other S100s, by the way. TNF alpha goes up. Here this is the hyperreactivity. This is upside down, meaning the lower numbers of inhalations of methacholine you need, the higher the, hyperreactivity. That means higher S100A9 is associated with worse hyperreactivity. Again, a correlation between the RAGE activation and hyperreactive airways. This is just to show you how complicated this is and how I really don't understand anything about it.
This is sort of a cartoon that was put together from all the inflammatory pathways we know that are activated in asthma. What you can see, you can point out all these RAGE agonists right through this. In these pathways, RAGE agonists and therefore the perpetuation of inflammation is basically given through these known inflammatory pathways. Making it up just a little bit simpler, this is RAGE. We believe it's really upstream of all these T2 high but also T2 low asthmatic responses. Now, we don't have good pharmacological treatment, but we can use knockout mice and Erik already showed some of that. When you look here at knockout mice that were stimulated with allergens. Again, most of these animal models are really allergic asthmatics, right? It's very difficult to do a T2 low model.
I'll show an attempt at that. When you compare that to the wild type, there is a huge blunting of IL-33, other cytokines with Alternaria, other models. Every time you take RAGE out with a knockout model, you will actually reduce the cytokine level that are typically activated in these allergic responses. The same is true here. This is a little bit more complicated because TLR4 can play a role in this allergic asthma as well. When you look at the responses from IL-25, IL-13, here is the famous TSLP that Mario talked about and IL-1α, you can see that RAGE knockout will decrease, not completely blunt, but will decrease these responses basically at relevant time points of all these cytokines. It doesn't need TLR4 knockout either. It is seemingly upstream. What you see here, it's not only upstream in this cartoon.
As long as RAGE is there, that's IL-13 activation. You see STAT6 can be sustained, meaning it is activated in a sustained fashion. If you take RAGE out, there is no permanent activation. RAGE seems to be important to actually sustain the inflammation in these airway diseases. It's not only to initiate it. This is seen here. Again, you treat with IL-4, for instance, in a RAGE knockout mouse, you don't see the eosinophils. p-STAT6, phosphorylated STAT6 activation, this is IL-5 and IL-13, same event. Using these cytokines to create an allergic model, a T2-high model, if you knock out RAGE, it is blunted or eliminated. Now T2-low, that's a hard one.
The only thing we found is if you have cigarette smoke exposure, so it's not typical asthma, but you know, again, an attempt to do a T2 low model, you're gonna see hyperreactivity that is actually eliminated by RAGE knockout, and you see neutrophils decreasing and a relevant cytokine decreasing as well. This is not a perfect model, but again, it suggests that there is an avenue to potentially approach T2 low inflammation as well. Now, these are the current guidelines by the NIH and the different societies how to treat asthma. Looks pretty simple and straightforward, and that has been true for about 20 or 25 years. Interestingly, this was published in 2020. Like there's this little box down here, and it says, "Consider adding asthma biologics." You know, not clear in 2020. That was two years ago.
Yes, they're lagging behind. If you go a little bit further into this is just another depiction of what Mario already showed. Well, there are these biologics, right? They're really targeting very, very specific pathways. To select the patients that will benefit from them becomes therefore somewhat complicated. I guess this box explains that. They didn't really wanna go into the complications of who will benefit or not. This is the algorithm that they think you should follow. Now, if you see a complicated algorithm like this, then you know this is gonna be very difficult to actually do. I guess we have to all send our patients to Mario to make the decision here, which you know, blocker you need and which biologic.
This usually translates that a lot of our patients will not get correctly treated or, optimally treated, I would say, because it's not simple to make these decisions. One of the biologics, I should have given that to Mario too because he was the first author on that paper, right, in the New England Journal of Medicine. That was one of the phase III clinical trials for Dupilumab. You can't read this, so I blow it up here a little bit. To take then a biologic and bring it into the asthmatic population, even though some selection occurs, you need to get a lot of patients enrolled to actually show positive outcomes that as depicted here. If we have a more broad inhibitor of inflammation, that would reduce that significantly. I'm not a statistician.
I'm gonna tell you how many patients will have to be in this trial, but I'm gonna refer to Javier on that. That is the problem with more specific biologics. On the other hand, if you select the right people, and this is Dupilumab as well, if you do it right, then you can show significant benefits. This is in this large population, there was actually significant improvement in lung function. Yes, using the right population works with the biologics to actually pick the right population for these biologics. That is the very difficult point. Coming back to patient profile, you know, Mario had a patient that was on multiple medications and still just never got as good as he or she should. This is just a regular sort of presentation of a patient with asthma.
We're going the regular route, what the recommendations are. After all of this failed, you know, there was a biologic introduced. Did a little bit of something, decreased prednisone daily, but you know what that actually means, having prednisone every day for patients and their consequences of that. That biologic was stopped. Another biologic was tried, but it never really in the end controlled the patient. What are we doing with those? I believe there, or maybe even for other people, RAGE would be a great target to have. Why do we use RAGE? I think I walked through this every part here, that we have good evidence, at least in silencing models, that this is upstream of inflammation in general and therefore helpful.
It might be really also helpful in T2 low asthma, that there is no treatment at the present time. Beyond asthma, right, we talked about COPD, we talked about cystic fibrosis. All of those have inflammation that is related to RAGE, and therefore, these are diseases that can be targeted with RAGE inhibition as well. Now I don't know, that's the end of my talk, whether we have a break or whether Javier San Martin is gonna come and talk about the clinical development.
We're gonna take a break for about 15 minutes while they bring lunch out. Let's reconvene at 11:55 A.M. Eastern. Thank you. I will give it a minute so we can be ready. Okay, next up is Javier San Martin, our Chief Medical Officer, and he will talk about the clinical development for the new pulmonary assets.
All right. Thank you, Vince, and I hope you're enjoying your lunch and I'm not gonna be a huge interruption, but I think I have very interesting stuff to share with you. I will present our initial thinking on the clinical development program for both ARO-RAGE and ARO-MUC5AC. Focus maybe on the first phase studies that are about to start very soon. But also I wanted to share with you our current thinking for the overall drug development, for both molecules, thinking about different indications and different path to regulatory filings. In every single drug or disease that I've been working over the last 25 years, I always was interested in the history of those diseases and the drugs that were developed to treat them.
With asthma, it's very interesting this disease was described over 150 years ago, and the focus was always the bronchoconstriction and the intermittent aspect of this disease. Patients get worse and then improve. That was kind of the concept of how this disease was known by. The first treatment, of course, was the ability to reduce the bronchoconstriction with beta two, and this started maybe 40 or 50 years ago. Over the years, they improved the beta two. They make it long duration in order to improve compliance, and to some degree, that approach took care of the bronchoconstriction part of the disease. It wasn't until about mid-1980s where the inflammation was considered a key component of the disease and perhaps the cause of the bronchoconstriction, and in fact, now we know that that's true.
Inflammation at that point in the 1960s and 1970s was treated with glucocorticoids and initially was treated with systemic corticosteroids. Of course, as you know, that is not sustainable long-term at high doses, so the industry developed inhaled steroids, and that was a significant improvement in terms of care. Of course, not all patients respond to it, and compliance has been always an issue with daily or twice-daily dose of inhaled steroids. About, like I said, 30 years ago, inflammation was a big deal, and it wasn't until about 20 years ago that we had the first biologic treatment. It was Xolair. Then another 10 years really without any other significant new biologic treatment.
The last 10 years or so, different companies start to identify right targets that you can block with antibody therapeutics, and that's the new revolution of biologic treatment in severe asthma, something that happened in rheumatoid arthritis, for example, 30 years ago. It's very interesting moment, I think, in asthma drug development. We just went to the ATS meeting last week, and I was really impressed with the energy, with the excitement around all this biologic, the clinical trials, the results, the subgroup analysis, the very good combined work between the academic group and the industry. I did feel that energy when a field is progressing to the next step.
That's why I think we're in the right time to initiate the clinical development program for these two molecules because the field is ready to keep learning and keep working on it. Again, one of the point is it was about 40 years since people recognized inflammation is a key component of the disease, and proper treatment was developed for it. Now mucus. Mucus has been described as part of the symptom and the phenotype in asthma, in COPD, and many other mucobstructive lung diseases. It wasn't until recently that was considered part of the problem, not just a symptom of evidence of the problem, but actually part of the underlying pathophysiology mechanism that increase the severity and the risk of asthma. That's very interesting because that was really in the last three or four years.
Here we are, three or four years later, developing the first target therapy to address this component of the underlying pathophysiology in asthma. Here, big picture how we're thinking about the two molecules that we will develop. One is ARO-MUC5AC, and of course, this is a new class of drug. It will block the production of mucus, and by that mechanism, hopefully will improve airflow and decrease exacerbations. Also, another concept that is relatively new is that mucus is not just a consequence of inflammation, but also is a cause of inflammation. Again, that's a very new concept and of course, one of the reason why we're developing this particular target.
The next concept with regard to MUC5AC is that, yes, the number one target will be severe asthma 'cause it makes a lot of sense because it follows the pathophysiology and where the pathophysiology meets the mechanical function of the drug. It also will allow us to study this drug in many other different mucostructural disease, including the asthma that is not well-served today with the anti-inflammatory therapies. On the other hand, we want to target inflammation, and we do want to develop the best possible anti-inflammatory therapy, and we think that will be RAGE. Why?
For everything that's been said before, Matthias really explained very well the role of RAGE with regard to the inflammatory response that you see in asthma is upstream of essentially everything else, but also may affect or intervene in the non-type two high, which is another very interesting concept. Maybe by being such a high level inflammatory cascade, it may prevent the fact that patients sometimes need to switch from one treatment to the other. That is the example of the patient that Matthias presented today. This broad class of anti-inflammatory, plus this is a drug that would be given in an inhaled as opposed to systemic, likely monthly, perhaps every three months. Again, back to a typical common problem in chronic diseases is adherence or compliance with treatment.
The fact that we will space this month apart, it hopefully will translate to improvement in adherence compliance, and that usually translate into improvement in clinical outcomes. We start with RAGE, and of course, this is the drug that we have to address inflammation in severe asthma. Airway inflammation is at the front of all the rest of the pathophysiology process of asthma. Inflammation increase mucus, inflammation, of course, affect muscle contraction. The idea is you block inflammation and you improve airway flow. Improve airway flow is the goal of therapy here. It should translate into improvement in FEV1 functional capacity symptoms and hopefully prevent exacerbations. This all has been presented today by my colleague, Erik, and also by Matthias, which is the very significant level of evidence that exists to justify this as a very good target.
There is studies that look at association of expression of this gene or increasing concentration of the protein. In both cases is correlation or associated with severe asthma, so that's one piece of information. The other piece of information is all we learn from the knockout mice models. Again, this has been described today. As a drug developer person, when I see that we have drugs or antibodies that essentially target each of these cytokines that are downstream of RAGE, I get more and more excited about RAGE being the right target that may take care of many of this. I have to say, people know about RAGE for a while, but of course, it's not a druggable target with a systemic treatment like an antibody.
We believe that this is the best, so far, the only way to address RAGE. Again, addressing RAGE cascade down, we're gonna tackle most of the cytokines and alarmins that are responsible of the pro-inflammatory process in patient with asthma. Also, the RAGE knockout mice provide some evidence that can also be effective in preventing inflammatory response in those with type two low asthma as well. Regardless what is the initial inflammatory pathway, being more on the type two high, eosinophil-driven or more like neutrophil-driven, it seems to be that blocking RAGE will improve or might improve in both type of circumstances. Finally, Erik shows what happen when you do the experiment by inhibit RAGE using an RNAi such as RAGE that definitely recapitulates all the stuff that we learn in the knockout mice.
This is I think a huge level of evidence that, as someone working in drug development, very happy to work with from now and take this to the clinic. Dr. Castro already presented some of the issues that we had today with regard to the treatment of asthma. We have much better treatment. In the last 10 years it's been this biologic revolution, but there is a lot of room for improvement. When I think about it, I've been doing the parallelism between the asthma biologic revolution compared with the rheumatoid arthritis biologic revolution 30 years ago, and at the beginning there was three drugs in RA: Remicade, Enbrel, and Humira, and people thought, "Well, that's it." Today there are 15 biologics with indication to treat rheumatoid arthritis plus another number of small molecules that follow similar pathway.
I think there is room for a lot more better treatment for asthma and also different patients may require different approaches in terms of administration and in terms of the specific type of phenotype of disease that they have. As I said before, the type 2 low asthma is another opportunity that may be this target, this drug will solve. Again, systemic subcutaneous administration, some people may prefer that, but inhaled or infrequent nebulizations could be a very attractive approach, that again, the goal will be to enhance adherence, compliance and by that means a clinical outcome. All right, let's jump into the clinical trial. This is the schema of the phase I clinical trial for ARO-RAGE.
Number one goal is assess safety, and we're gonna assess safety by, of course, collecting AEs, adverse event, serious adverse event, laboratory parameters and FEV1 or spirometry including DLCO. We have a very robust safety assessment included in this study. The other goal, from the therapeutic perspective, and the more important is the target engagement. It's really the ability to describe the pharmacodynamic and we're lucky that in with this target, we can do that in a non-invasive fashion. I will say a few more things about that later. The way that we study the patients and the normal healthy volunteers, the normal health is particularly we have a bronchoscopy at baseline and another one at week four.
Patients will receive one dose of ARO-RAGE, and the reason that we'll receive one dose is because the duration of effect is expected to be really long, so we don't see the need to do multiple dose in the normal healthy volunteer patients. Then we collect sputum almost weekly at the beginning and then bi-weekly. We will enroll the normal healthies in four different cohorts with ascending dose from 10, 20, 40, and 80. When we get to the 40 mg cohort, that cohort after it's complete, and we will enroll eight people in each cohort, six in active, two in placebo, that cohort will enable, after the 22-day safety assessment by the DSMB, the asthma cohort. The patient with asthma will have a broad range of severity of disease.
We decided to go with patients with GINA 1-4 that will facilitate the enrollment. We will ask for patients with type 2 high. We're not going to the 300 eosinophil, because we think that 200 is good enough. With the intention to say this patient with this phenotype will represent the type 2 high and will be relatively easy to enroll. The patient with asthma will be dosed twice, at baseline and at day 29, and we will follow them for 113 days. We're gonna enroll eight patients, six of them will receive ARO-RAGE, and two will receive placebo. You can see the doses here. The maximal dose will be 80 mg, and this dose represents the dose loaded into the nebulizer.
Another reason to be excited about this particular program is that we have the ability to measure a biomarker that will speak to the pharmacodynamic effect of the drug in a non-invasive fashion. We're collecting, as I said, sputum in both normal healthy volunteers and patients, serum of course in everybody and in the normal healthy we're also doing a bronchoalveolar lavage assessment. In all these three specimens, we're gonna measure soluble RAGE. Of course, important will be to correlate all these measurements because the goal is eventually to have just serum RAGE as a soluble RAGE as the biomarker that we can easily use in large-scale studies such as the phase II, where we're gonna do the dose selection or even the phase III.
It's one of those things that when you fast-forward, it might even help clinician to select what's the best patient for one therapy or the other. In the normal healthy volunteer, we're also, when we do the bronchoscopies, we have bronchial brushing, and that will be used to measure RAGE mRNA expression. I think the combination of these four parameters that assessing RAGE will give us a good idea of how to assess pharmacodynamic effect in this condition. I think this is one of the key benefit of this particular program, and we did not have this this opportunity with ANAC. This is something that we welcome and from the drug development perspective, I think it's adding a lot of value.
I just wanted to show an example of one successful drug development in severe asthma, which is the Dupilumab example, to give an idea of how many patients, how many phases you need to go through to go to the FDA and file this drug. This is a good example of a regulatory path forward. In the case of severe asthma, that is very well established. This after the phase I studies in normal healthy, it was PK and PD, they did the first study in severe asthma, the phase II/b study in patient with moderate severe asthma and with T2 high. They only enrolled 104 patients. It was a relatively short study of 12 weeks. They did this approach about taper standard of care, and when the taper is done, then evaluate exacerbation and pulmonary function.
That's a clever approach to do that because allows you to have fewer patients. This was also done recently, I think it was in the anti-IL-23 program that was published in New England a few months ago. That, that's a way to enrich the population when you are at the beginning. Get the patients that are more likely to respond, titrate to high, and create a protocol design that will prompt them to have exacerbations and eventually demonstrate the efficacy. I thought that was a very smart approach for the first study that achieve a proof of concept, but not necessarily a dose selection. In order to go to dose selection, they needed to do a phase II/b study, and this was a much larger experiment with 269 patients, moderate severe asthma and any level of eosinophils.
Broader the patient population with regard to the type of disease, but still moderate severe asthma, which is the target population for which this drug could be used. Endpoints in this type of study, again, you cannot be very creative. FEV1 is a classic endpoint that display the improvement in airway flow. Annualized rates of exacerbation, of course, is the key approvable endpoint. It's important that in this field you start to see those endpoints as early as phase II. Of course you need phase II studies of this magnitude over 700 people.
From there, they move over to the phase III study, and here again, patient with severe asthma or moderate and severe asthma, any comment with regard to the type of asthma, and that I think it was that drew the very large sample size of 1,900 patients because I'm not a statistician either, Matthias, but I'm very sure that they power the study using an assumption for an effect size on the type II, knowing that the type 2 low may not contribute too much to the effect size. Therefore, I think they had to do this type of design, which is perfectly fine because in the end you want a broad label, and this is the way you try to do a study to be able to have a label that is broad and include patients with different type or different phenotypes.
Again, the endpoints are very much the same. The last registration study has to do with patients that, of course, severe asthma, that they were receiving oral steroids. It's a sizable number of patients that still receive oral steroids. The idea is, can you treat this patient with a more effective anti-inflammatory and taper that oral corticosteroid therapy? Because of course, oral corticosteroid therapy has a lot of consequences, that you don't want to see. This is, you know, this is a guidance. The tezepelumab program is very similar, maybe a couple of different features, but generally speaking, this is how the FDA expect you to show up when you file a treatment for asthma. This is our initial thinking in our phase II study. We will include patient with a high and low. We want to be inclusive, but why?
Because we think that this molecule actually has the capacity to be effective in both type of asthma. The endpoints will be the one that most people use, exacerbation, FEV1, symptoms of course. The goal of the study will be dose selection. This will be a phase II/b study. We don't know still the size of the study, but it will be to enable a phase III study. A phase III study now, I think it will be again, patient with severe, moderate or severe asthma. Those are the one that needs more help. We will be inclusive of the different type of asthma more likely. Again, the endpoints will be similar to what has been done so far.
We think that this therapeutic ARO-RAGE could play a role in COPD, could play a role in cystic fibrosis, and we will start to consider how to move from severe asthma to COPD. As you know, Dupilumab is now running a study in phase III. I think the concept of a better than glucocorticoids anti-inflammatory could be helpful in COPD. I think it's out there. People believe, people are working on it. We may need to consider whether we need a phase II in COPD, what we need to know to have the confidence to run a large phase III COPD study, because that will be a large study and clinical outcomes will be critical for approval. All right, let me switch over to MUC5AC. Airway obstruction is what, in the end, causes disease.
Airway obstruction is what cause symptoms. Airway obstruction is what cause exacerbations. Therefore, it's at the center of the disease. What I think is interesting, again, as I said before, is the idea that mucus is part of the mucus obstruction is relatively new as a therapeutic target. It's not just because there is a mechanical obstruction component, as Dr. Castro show in those CT scans, but also the excess of mucus increase inflammation. This is a cycle and applies to both drugs. You know, when it's inflammation, increase mucus, both together impair mucociliary and airway function. When you have mucus drive inflammation as well.
The way I see this is you can attack this problem one way or the other, and I hope at one point we will be able to distinguish who are the patients that might benefit more from one or the other. Again, all this has been said already, but I think it's impressive the level of evidence that we have from observations with regard to the increased expression or concentration of MUC5AC and severity of the disease, severity of the numbers of mucus plugs that you observe, and exacerbation. The three components of the disease that you really care about, they're all in a way drive by the increase in MUC5AC that you see in patients with both severe asthma and COPD as well, and other diseases that I will comment on it later.
In the case of MUC5AC, again, already was presented by Dr. Castro, there is a clear association discovery in more than one GWAS study. That level of evidence add to the evidence to justify this target. The knockout mice again replicate the same concept. Once again, Erik showed that when we do the experiments, we replicate again the same concept that is able to decrease airway resistance. We hit every single level of observation and preclinical evidence to justify these targets. Dr. Castro very nicely illustrate this concept, which is relatively new, the idea to show the segments of the lung that is completely obstructed by this mucus plug. It's so interesting to say that correlate with disease severity, and it can be quantified easily.
We're thinking about this as, number one, a tool to identify patients that may be tailored to this given therapy, but it may be it's an endpoint as well, and we can incorporate that into our phase II studies and perhaps see whether there is a decrease in number of segments that are mucus plug or preservation or prevention of new mucus plugs. This is new stuff. It's developing as we speak, but I think it's another opportunity to have another biomarker or surrogate to learn more about how this drug affects the disease and the underlying pathophysiology problem. As he showed before, normal lungs have no mucus plug whatsoever, and patients with asthma have a large proportion of them, a substantial number of mucus plug. Here, I think the correlation between this and severe disease is very clear.
Again, when you think about MUC5AC, seems to be a very, very appropriate target to address these issues. Phase I study with MUC5AC, safety is the first priority and the first assessment, and we're gonna do that exactly the same way as we do it in the RAGE study, which is clinical, laboratory, and spirometry. Here we start with a normal healthy single dose or ascending single dose, in which you can see 24 mg, 56 mg, and 108 mg, one single dose. Once we're done with the first cohort, that will enable the multiple dose normal healthy volunteers cohort. The same will happen with the next cohort of 56 mg, going from single to multiple, and at the same time with patients. Importantly, in this case, we selected moderate and severe patients with asthma, with severe asthma.
You can see their airway obstruction 40%-80%. We're looking here at more severe patients to really be able to see the signal of MUC5AC reduction. Part of the biomarkers assessment will be sputum. As you can see, this is in black. We're asking the patient to do this induced sputum very frequently, weekly or biweekly. Also the normal healthy volunteers in the multiple dose will have a baseline bronchoscopy and a post-dose approximately one or two weeks after the third dose. The multiple dose cohort for normal and patients will be three doses separated by two weeks each, and then the bronchoscopy is a week or two later. The total duration of the study will be approximately 85 days. How we envision the long-term clinical development program for this molecule.
Moderate and severe asthma, again, the pathway is being written, but I think we have a couple of new onset here, particularly regarding to the CT scan and the perhaps the ability to select patient based on the number of mucus obstruction in order to understand whether we can see an effect there or no, or that per se select the population that more likely will response to this therapeutic. Because it's reasonable to think that those patients who have more mucus obstruction or mucus plug are those who have more mucus in general. Mucus is a cause of obstruction, and mucus is a cause of inflammation. Again, this is very interesting within the drug development process. At the same time, the academicians and the science is moving forward the same concept, and we're both learning together.
I've been in that place before, and I think it's very different when you are working in that synergistic approach with the academic and the science. Eventually, we'll do a phase III moderate and severe asthma study. Again, the details, how we're gonna go about, what will be the patient population is something that we need to learn more as we go along. It's very important for this target that we're thinking in a number of other diseases where the mucus obstruction is the key feature. One of them is COPD, of course. Then cystic fibrosis perhaps is another opportunity, non-CF bronchiectasis, and primary ciliary dyskinesia, and I will say a few things about each of these. This slide was shown before, but just conceptually again to say, you know, normal mucus layer is important.
It's important to enable the ciliary movement to get the mucus out of the system. When you have too much of the and a sticky type of mucus, then it makes the work of the cilia a lot more difficult. When you think about a disease like PCD, which are patients who have the highest concentration of mucin, and they already have an issue with the mechanism by which they get rid of the mucus, then it seems to me that this is a very incredible target. Now, it's a very rare disease, but I'll make a few comments about that later. I see this as a great opportunity. CF is a similar issue, mucociliary clearance impair.
Now, some patients are doing really well with the current therapy, but, you know, the new normal patient had no choices, and they have a lot of mucus production, mucus stasis. Mucus stasis increase the risk of infection, and you know the cascade of clinical events in those cases. Non-CF bronchiectasis, same deal. When you think about those patients, it's all about expectoration, sputum, phlegm, and very symptomatic diseases. COPD, particularly in the bronchitis subset or subtype of COPD, one of the key problem is the accumulation of mucus and how that impact quality of life. This is how we see COPD and why I'm really excited about this. One is that I wanted to contrast the picture I saw at the American Thoracic Society meeting last week between asthma and COPD.
I went to a number of presentations, particularly some of the industry seminars and symposium. In the severe asthma, it was all excitement and subgroup analysis, all kind of interesting stuff. When I went to the COPD, it was about compliance and about who can do a longer acting beta to longer acting LAMA, longer acting inhaled corticosteroid, how you can combine in one puff, so make it easier for the patient, which is definitely important. And don't get me wrong, I work mainly in chronic diseases, and regardless of the disease, compliance is a problem and is a real issue. Make it more friendly. The current therapy is a good idea.
Really, it wasn't any news that I was like, "Okay, the COPD field is moving this direction to take it to the next level." This is my area of expertise as a clinician, but that was 25 years ago, and I did not learn anything I didn't know already. It is a huge opportunity when you see a target that may address an underlying problem of the disease. I think it's one of those critical moments in drug development. It's a disease with a lot of morbidities, as you probably all know, and all those morbidities are easily assessed and quantified clinically. The development program for a drug in COPD is relatively straightforward. A lot we need to learn to select the right population, to do different correlations, and so forth.
I'm really very excited about this opportunity. One of the reason is this. These are three different PRO instruments that people use in clinical trials in COPD, and they're all driven by the burden of mucus in these patients or the clinical daily living quality of life impact of the excess in mucus. I see this an opportunity not only to hopefully improve pulmonary function, prevent exacerbation and hospitalization, but also to really improve the quality of life. It looks like the PROs are tailored to really investigate how much that mucus hypersecretion translate into clinical event that impair these patients' quality of life. This is from the drug development perspective, it's really a good place to be, to say you are addressing an issue that you can clearly measure and impact the quality of life of patients.
Last, I would say there is no treatment that prolong survival in COPD for the most part, perhaps oxygen. Imagine if this changed the underlying condition. It is a disease-modifying agent, and thus prolong survival. I'm dreaming here, but I will not exclude that notion that as we progress in drug development, one day we start to study the really severe patients that had a phenotype that this intervention might be helpful. This is very preliminary, but you know, this is how we see the COPD clinical development. Perhaps the new stuff here is the CT scan. Again, back to Dr. Castro presentation. CT scan today is now part of the normal evaluation of a patient with COPD. Why? Because there is nothing to do about it.
You know, depending on the phenotype, there is not really a lot of different decision-making as a clinician. Now, if you have a therapy that specifically target a component of the disease, you can enrich the patient population and perhaps affect the disease a lot more. This will be the first time that in COPD, despite for many years, people know that there was a more emphysema phenotype or bronchitis phenotype. Now we can think about there is a tool to identify that population better, and there is a potential treatment that will address the underlying condition. Right. I also have some background in rare disease, and I get very excited when I think about this molecule and these three conditions. I already mentioned primary ciliary dyskinesia. The treatment is antibiotics, airway hydration, bronchodilators, so very much symptomatic treatment.
3,000 patients in the U.S., so that give us the opportunity for an orphan application. The mucus is at the center of this disease. The mucus stasis is what increase the risk of infection. Infection produce more inflammation more mucus. Mucus make the cilia more difficult to really do what they need to do. The idea to reduce mucus in this disease, it seems to me a really interesting approach. Non-CF bronchiectasis from the clinical development will be challenging because it's a very heterogeneous population, but it's not small. Again, this is a condition where mucus plays a fundamental role in the daily living, in the quality of life of this patient. When you have that connection, it's also a very important aspect to develop a drug.
Finally, as I said before, cystic fibrosis, we think that there is an opportunity there. Again, mucus stasis, impaired mucociliary clearance is what drive the disease. Of course, there are a number of patients with a new treatment that are doing much better, but still there are room for improvement, and there still are population that the new CFTR are not able to address. So we're considering this as well. To conclude, you know, we're developing two molecules, MUC5AC with a complete novel mechanism of action, having a potential new tool to identify patients that might benefit from it and that is a unique benefit in a clinical development program. That can give us a broad pipeline. This is a molecule with a pipeline itself, and I think that is very unique.
ARO-RAGE, our vision is hopefully to be the best anti-inflammatory therapy. Again, local administration, non-systemic administration, very long intervals between doses, and that is in itself a benefit. Again, a broad pipeline because hopefully it can be used with both types of inflammatory asthma and perhaps with other conditions such as COPD as well. As a drug developer, it doesn't get any better to be in a place like this. The idea to develop in parallel two drugs for the same disease is a unique opportunity. We will need to be really creative to differentiate these clinical programs, to eventually differentiate this product the day that it sees the market. That in itself is a very interesting concept. The same applies to COPD.
We have the two molecules, so the two pathway, MUC5AC and RAGE, that we could develop for this disease. Now, these two will follow a relatively well-known regulatory path, and it will take time. At the same time, I do believe and we do believe that these drugs may have other use and opportunities in rare diseases, and that could be something that we'll do in parallel. The regulatory clinical development will really be divided into the large indications and the smaller indications in which we will need to be creative to move this really fast into the development program. I will pause here. This is like a very special moment for us, and I want now to invite Anjali Sharma to provide an initial reaction to how we're thinking about the market and these molecules. Thank you.
Thank you. Everyone can see me. As my colleagues have mentioned, ARO-RAGE has the potential to target underlying inflammation, while ARO-MUC5AC has the potential to target mucus obstruction. A number of pulmonary diseases are addressable with these mechanisms of action, from broad indications like asthma and COPD to rare diseases such as cystic fibrosis and primary ciliary dyskinesia. For today, I will focus on the unmet need and potential of these products in asthma and COPD. Currently, approximately 25 million patients are diagnosed with asthma. The funnel on the left represents patient segmentation by the Global Initiative for Asthma guidelines, which categorize patients based on the level of therapy needed to control their asthma. Patients start on inhaled corticosteroids and step through add-on LABA and LAMA therapies. It is estimated that there are roughly 6 million patients on steps four and five whose disease is considered moderate to severe.
These patients are uncontrolled due to lack of efficacy and the need for daily compliance with inhaler use. Of the patients who are uncontrolled, 1 million are considered to have truly severe asthma. Currently, biologics are indicated for these patients but have low penetration due to the need for subcutaneous administration, access and reimbursement restrictions, and efficacy issues. Additionally, TH2 low patients represent an underserved population. I will go into the unmet need for severe asthma patients on the next slide. In June of last year, we surveyed almost 40 pulmonologists and allergists regarding the unmet need experienced in both severe asthma and COPD. The right-hand side of this slide indicates some quotes we heard. Physicians expressed the need for therapies that are convenient for patients to use or that will reduce the need for daily therapy. They also identified non-TH2 patients as an underserved patient population.
They told us they are looking for therapies that can help address the underlying cause of asthma and not just treat symptoms. The story is very similar in COPD. There is a large patient population that is diagnosed with COPD who continue to experience low FEV1. The funnel on the right segments patients according to the Global Initiative for Chronic Obstructive Lung Disease or GOLD guidelines. There are almost 7 million moderate to severe COPD patients with FEV1 below 80%. Uncontrolled COPD patients continue to experience multiple annual exacerbations and emergency room visits, putting stress on the healthcare system in regards to costs and resources. When discussing the unmet needs for these patients, allergists and pulmonologists echo similar sentiments as they do for severe asthma.
The left of this slide indicates pulmonologists reported challenges in COPD and their ranking for importance in solving those challenges on a scale of one to 10. The right-hand side are quotes from our market research. Physicians are looking for therapies that can slow the progression of COPD and not just treat symptoms. They also believe that there is a major inflammatory component of COPD that is not being addressed by current therapies. They described the daily burden of therapies, especially on COPD patients who may have cognitive and dexterity issues or low inspiratory rates. It is clear that current therapies are not adequately meeting the needs of asthma and COPD patients or their physicians. ARO-RAGE has the potential to be a better anti-inflammatory that can work upstream in the inflammation cycle to inhibit cytokines. ARO-MUC5AC has the potential to be a first-in-class therapy targeting mucus depletion and mucus obstruction.
This is an underlying component of many diseases, including asthma and COPD. We look forward to exploring these therapies in the clinic and assessing their ability to reduce exacerbations and improve airflow, thereby addressing underlying disease progression and improving patient quality of life. These therapies also have the potential to offer biologic-like targeting and effectiveness with the convenience of a less frequently dosed inhalation therapy.
This can lead to improved patient acceptance, compliance, and ultimately better disease control. We are excited for the potential of our pulmonary portfolio to address a broad range of lung diseases by targeting a variety of mechanisms of action. As new targets are identified, we are continuously prioritizing indications based on unmet need, market opportunity, and the scope of clinical development required, among other factors, in order to approach the development of our pipeline thoughtfully. We look forward to keeping you updated on our progress. I will turn it over to Chris.
Do you want me to move this on to the way? Move it that way. Okay. That was a long day. Thank you, everyone. Thank you to all the speakers. I think that was a really helpful overview of where we are and what we're thinking. Of course, thank you to our guests, Mario and Matthias, you know, for increasing the IQ of the room by an order of magnitude or so. This slide, Anjali's last slide, is a really good one. I was gonna steal it, but I didn't. It's just such a nice representation of how many people we can help, I think, with just these first few programs, and we're so excited about this. This, you know, this is just for the fit few ARO-RAGE, ARO-MUC5AC, MMP7, and then, of course, respiratory viruses.
You know, we'll see where that goes. When we were going through our slides last night, someone from our team had said, "You know, that's a whole company," and it's right. In fact, that's a whole company and not an average biotech company. That's a whole broad and diverse and deep biotech company. That's only one part of what we're doing. In any event, I digress. When I introduced the presentation earlier, I talked about this R&D day we had about four and a half years ago. At that time, we had zero clinical programs. Zero. Now fast-forward to now, by the end of this year, I expect that we're gonna have 13 clinical candidates in clinical studies. Seven wholly owned, six partnered.
By any measure, that is a successful track record over the last four and a half years. When you are handicapping our chances of success for this next platform, this next franchise, think of that track record. This is our pipeline right now, spanning cardiometabolic disease, burgeoning pulmonary division, of course, the cadre of liver diseases, and we think also by the end of the year, possibly, our first muscle candidate in the clinic. Over the past four and a half years since introducing the TRiM platform, we've built what I see as a robust and scalable and substantially de-risked hepatocyte-directed set of drug candidates.
This has provided, we think, an enormous amount of hope for millions of patients and has created a substantial amount of value for us. I think that we are on the cusp of doing that same thing for pulmonary. I look forward to keeping you updated on our data this year and throughout the life of this franchise. Thank you again for coming. I guess we have questions. Is that how it goes? Go to one more. Here we go. Q&A session. All right. Do I want the speakers to come up here to answer questions? Let me rephrase that. May I have the speakers come up here to answer questions.
We have a couple handheld mics also.
All right.
If you want to bring up Mario and Matthias, go up on the podium.
Ted, you.
Great. Thank you. Can you hear me okay? Ted Tenthoff, Piper Sandler. Firstly, thank you. This was really informative. One of the things that I walked away with, I really appreciate the value, potential value of soluble RAGE, but also as potential biomarkers. I guess firstly for the doctors, do you see the opportunity of a nebulized therapy that targets upstream of select cytokines likely to be used ahead of the biologics, almost in a similar case as what we've seen with Otezla and the orals in RA? That's the first question. I guess related to that, is there a higher safety bar for that kind of profile and/or maybe even a lower efficacy bar?
I don't know if that makes sense, but I'm wondering whether you see the potential, and obviously this is gonna be data dependent, and we're still very early here, but do you see these potentially being used ahead of biologics?
I can answer first then Matthias can add to it. When we think about the spectrum, at least in terms of asthma, and we can also address it on COPD, there is this gap right now between inhalers and biologics. I was part of drug development for Fevipiprant, which was an oral agent blocking prostaglandin D2, and it failed in its primary endpoint, and therefore was not taking forth. That was a perfect example of an oral drug that would be a predecessor to phase or to biologics, stepping up to biologics. In the pulmonary world, the allergy world, that takes care of the vast majority of asthmatics, we are very comfortable with inhalers and nebulizers. Our patients are very comfortable with inhalers and neb. They use them every day.
It's a very easy segue to move to a nebulized treatment like this. The jump to subcutaneous therapy or intravenous therapy has not been as easy. Certainly, that requires patients to come into the office for their initial injections. The field has now transitioned to self-injection, which has helped, but still there's a pretty substantial gap in patients that are uncontrolled asthmatics that, you know, we just wish we had one more kind of step up before we stepped up to an injectable therapy.
I would support everything that he said about that. I mean, it seems easy to inject somebody, right? The truth is most patients don't want to do that. The question then is inhaled therapy, they're used to this, most of these patients, right? We're not treating people who have never had an inhaled therapy. That is easy to do. The distribution of the ventilation will be interesting to study and, you know, see how that goes. Is there a higher safety? You asked, is there a higher safety.
Higher safety, lower efficacy.
Yeah. Lower efficacy, I can't speak. I'm not the FDA. You know, this is at the end, the regulators that, like, make that decision. In terms of safety, the worry, and I'm not saying that is a terrible worry, but you know, if you block a lot of the inflammation, is there still sufficient beneficial inflammation if you need it? The answer to that question is I think we will find out. If you look at, for instance, RAGE knockout mice, again, this is mouse, it's not humans. If you look at knockout mice, they can deal with pneumonias, et cetera, just fine. It seems that if you have TLR4 still working, you get the initial inflammation, and that will resolve the problem with the infection, but you don't get the prolonged, not so good inflammation. At least from that data set, I don't believe that there is a higher level of safety, but because these things have been looked at.
Okay. I guess kind of following up on that a little bit, and this is sort of a conceptual question, but with respect to the dynamics around ARO-MUC5AC, you know, how is this working? Does it just kinda hang around there and then really silences the MUC5AC when a trigger causes it to be overexpressed? Is that the way to think about it? Or is it really constitutive knockdown, and it remains suppressed during a trigger and an overexpression episode? Thank you.
Well, you need to look at two different situations, right? There is the situation where MUC5AC has not been overexpressed as yet, and there you would let the trigger sit there. I mean, there is evidence that the trigger sits there and will not allow MUC5AC to go up. If you already have a situation where you have a mucus plug, then that's another situation. There you need to get the trigger in the vicinity of those producing mucus cells, and then it will reduce the production. You still need to clear at one point in time that plug, right? Because this RNA does not do that. But there are techniques to at least help doing that, and that will be interesting to see how much of existing mucus plugs are removable. As Mario showed, there are even spontaneously remove. There is spontaneous removal of plugs in certain patients without doing much about that. The question is how does that go forward? That's the idea.
Yeah. I suspect clinically this goblet cell metaplasia that drives the mucus in our patients is an inducible phenotype, and that we can reverse by suppressing that mucin production. When I take a biopsy in my severe asthma patients, that they're just loaded. This is by taking systemic steroids. They're just loaded with goblet cells. The idea here is we can reverse that phenotype and suppress that MUC5AC production. We've had a lot of discussions about the mucus plug, and I ultimately think that the smaller airways are. The pathobiology there is primed for this mucus to form there, and that there's a fair amount of turnover that's going on. Even though those plugs persist, they're likely plugs that are forming and reforming over time, and that with this technology, I think we can reverse that process.
Thank you. Hi. Lisa Walter here, Senior Research Associate on Luca Issi's team at RBC Capital Markets. My first question is, maybe more so for Chris Anzalone. Are you ever going to show the human clinical data from the ENaC study? I believe you dosed about 24 normal healthy volunteers and four patients with cystic fibrosis. Will you share that at a medical meeting sometime this year or later on? Just wondering about that.
Yeah. We don't have plans of that right now. It's been difficult to interpret, to be honest, just given the complexity of interrogating that. We'll see. Right now, we don't have plans, but that may change.
Okay. Thank you. Just one more question. Just maybe more on your strategy here. You know, from following Arrowhead, the strategy in the past has always been to go after genetically validated targets or pharmacologically validated targets. With these lung programs here, this seems to be a little bit of a divergence of the strategy and maybe perhaps opening up yourselves to a little bit more clinical risk. Just wondering what kinda made you change your thinking here, and what made you kinda wanna go after these targets that don't have a well-defined, you know, human genetic knockout component that you can rely on?
I'm gonna defer to these guys. I'll tell you. I don't think it represents a departure from our strategy. Our strategy has always been to minimize target risk as much as we can. I think that these targets do that. You know, is there no target risk? Well, no, because no one has been able to, you know, to intervene in these ways. I think the evidence is compelling that should we safely and effectively knock down these targets, you know, positive phenotypes will result. Again, I'll defer to the experts on that. I you know.
At least in the common chronic lung diseases, we've been investing millions, probably billions of dollars in genetics, and we have no treatment based on genetics at this point, other than my colleagues' cystic fibrosis patients. It is a tough bar to, I think, at least in the pulmonary field, to do a genetically based targeting strategy. Matthias .
Well, I agree. What you also said, you know, even in the GWAS, there was MUC5AC coming up.
Yeah.
Which is actually interesting. I'm actually surprised about that. That's not necessarily what you genetically validate, and you see it that at COPD. If we're waiting for a genetically validated target, we'll not gonna get ever a treatment that changes COPD, for instance. I think the targets are very well validated through programs both clinically and pre-clinically, that show how especially MUC5AC but also RAGE play very central roles in mucus clogging and inflammation in the airway. I'm not sure exactly what genetic proof that would require, right? To say this is a good target. I think the preponderance of evidence shows that that's really important. The MUC5AC knockout mouse, for instance, shows that mucociliary clearance is established or continues, but these mucus plugs are gone.
It's not just a target of a mucus-producing cell because that will probably be detrimental. You know, you need mucociliary clearance to clean your lungs, and you need MUC5B, for instance, to be there 'cause otherwise you're not gonna do something good. Burton Dickey always says MUC5AC was , “invented” for catching the worms that come out of your gut and go through the lungs to come out. As carriers, basically. Catch them and let them be there. That has some reasoning that we , “have” MUC5AC, but in disease states like asthma, it's detrimental because you have these mucus plugs and in CF as well. I think it's validated, maybe not genetically, but the preponderance of evidence shows that this is really highly relevant targets.
Thank you.
Hi, good afternoon. This is Sahil Kazmi from B. Riley Securities. Thanks for taking our questions. First question is, can you comment a bit more on the relationship between MUC5AC and, MUC5B? Is this a cooperative or additive relationship? And does the knockdown on, you know, MUC5AC have an impact on MUC5B?
This is a highly complicated question. We think about mucins, especially the gel-forming mucins in the airways, specifically MUC5B and MUC5AC, as mixing in the airways and provide you with some sort of a layer of mucus that you can then remove. That is not as simple as it sounds because the major mucin that is under normal condition produced is the MUC5B, and that will maintain your mucociliary clearance. MUC5AC comes up in disease states, and there were some data shown. I mean, I don't know whether they were clear, but MUC5AC is that sticky mucus. Like, it's there to catch things. It's actually even tethered to the airway surface and therefore very difficult to remove. The MUC5AC is not a gel-forming mucin intended to remove things from the airway.
This seems to be more to hold things there. They don't perfectly mix. Genetically, they're not. You know, if you knock one down, the other one will not be knocked down. Now, can you have two MUC5B? I don't know. But this seems a mucin that you can then cough out, and do it better, whereas the MUC5AC is making these rafts not going anymore. I'm not sure that answered the question that you really had.
No, no, that's very helpful. Thank you. Very helpful context. Maybe just one more question in the context of what we saw in the ENaC program, acknowledging its early days in IPF, but we're seeing some pretty good durability out to day 42 initially. How long do you think that can go with a single dose?
You asking me? I have no answer to that.
Just upon if-
Sure. We can, and you're asking about the MMP7.
Correct.
Just in general?
Yes, exactly.
You know, I think, it's unclear in. We actually have a duration study ongoing right now in monkeys with that trigger. If you look at the RAGE data, I mean, I think that's kind of best case scenario where we're getting single dose at three months of effect.
Mm-hmm.
You know, maybe less so durability with something like ENaC, which was in the order of weeks. Call it somewhere probably better than ENaC and out to what we're seeing with RAGE. That probably gives you a way to handicap it, but we'll see. We'll know soon.
Excellent. Thank you.
Hi. This is Prakhar from Cantor. Firstly, for the KOLs, is there good evidence on what's the right cutoff for high and low levels of MUC5AC, similar to what we have with eosinophils? For the company, how do you plan to incorporate that strategy in your early phase I trial to get a signal on efficacy similar to what you're doing with RAGE, with high Th2? I have a follow-up. Thanks.
Do you wanna answer first, and then we'll answer the clinical question?
Well, I don't know if there's an absolute level because none of them have been clinically validated yet, what the optimal level is. I don't know if the company has any thoughts on that or...
Well, yeah. What is known is that the higher the severity, the higher the mucus plug, the higher the MUC5AC. I think I would go more from the clinical phenotype as opposed to the biologic to identify patients that might have more expression or overexpression, overproduction of the protein that we can probably affect. I think that's the beauty of that program, that you don't need to go to the detail. There is clinical features associated with higher expression of MUC5AC, and I think we're gonna go after those initially.
The ratio, right? The ratio of MUC5AC to MUC5B seems very important. You cannot just say, "I want that amount of MUC5AC." It's the ratio, and if you reduce that and you rely on the MUC5B, that's where the transport of mucin or mucus gel is. In that sense, the less of MUC5AC, the better.
Thanks. For the company, the number of doses, if I'm not mistaken, for the phase I trial were more in MUC5AC. Just curious if you would expect durability between the two targets.
Say that again.
The number of doses that's being given in the phase I trial.
It's three doses, two weeks apart. What was the other part of the question?
Would you expect differences in durability between MUC5AC and RAGE phase I?
Well, based on the preclinical data, yes. The RAGE is about three months and counting. For MUC5AC, it's more in the weeks, three to four weeks range. That's why we're dosing every two weeks, but one single dose. In RAGE, we're doing one dose, and we're gonna learn clinically how long it lasts.
Thank you.
Right. Yeah, we'll follow those patients out to day 113. Is that right?
Right. We follow for RAGE, we'll follow them beyond that. There's a threshold they have to come back to a certain level. I would also mention that, pre-clinically, RAGE is very easy to follow 'cause we have a serum-measurable biomarker. It's been a little more challenging to track duration with MUC5AC in animals, specifically in the monkeys. I think we'll have a better luck with that, easier to measure MUC5AC in the sputum in humans, even in healthy volunteers.
This is Madhu Kumar from Goldman Sachs. Thanks for taking our questions. I guess the first one's around dynamic range. This is really for both the company and the KOLs. What is the effective level of RAGE and MUC5AC knockdown that's needed to recapitulate complete genetic loss of function? And I guess the kind of corollary question to that is, do heterozygotes for RAGE or MUC5AC see kind of impacts in model systems to date?
We wanna start with the KOLs.
I am not aware of heterozygosity in MUC5AC or RAGE. I can't answer that question. I don't know the answer.
Don't either. Okay.
Yeah. As we showed, the preclinical models for MUC5AC, you know, we're triangulating on, we think we will need to achieve greater than 50% silencing for MUC5AC in order to have a protective benefit. We don't need to completely ablate it. For RAGE, the dynamic range is much higher. Right now there isn't a single threshold that we would say is necessary for an anti-inflammatory effect. We believe it's deeper than 50%, and we'll be aiming for as deep as possible because it's well-tolerated.
Okay, great. Second question relates to infections. You mentioned that certain types of pneumonia are not really affected by RAGE knockout, but other types of infections are. Like RSV infections are affected by RAGE deficiency. Gram-negative bacterial infections are affected by RAGE deficiency. At a kind of practical, kind of clinical development perspective, how do you think about kind of monitoring those types of infections in the real world as compared to in a laboratory, those animals aren't constantly exposed, hopefully, to RSV and gram-negative bacteria versus people in the real world are?
Do you wanna answer that first?
No.
No? Okay.
We didn't specifically think about that, but I would say something that to me clinically makes sense. These are chronic therapy. Patients will be treated chronically with the intention to prevent exacerbation. Exacerbation likely can be due to RSV. I don't see this as there is an acute event, let's treat this patient. I think this is like the biologic, like inhaled steroids. You modulate the immune system with RAGE. You modulate what happen with mucus with MUC5AC. When the insult comes, that lung will be ready to response more properly. That's the model of how we think this will work, because there is a bunch of model that shows improving or prevention on exacerbation, you need to have background therapy to do that.
I do think it's a good question, Madhu. I do envision that, you know, we're gonna have to carefully monitor these patients in the early phase II studies, to look for that as a possibility. Fortunately, they're pretty rare in my adult patients. You know, we see tons of RSV in infants, but we don't see it as often in our COPD population. I think fortunately, it's gonna be rare, and then the question is, you knock it down to a level that still sufficiently protects you, is the key question?
We saw this question coming up in other biologics, right?
Yeah.
I mean. This is not a unique one in terms of that. They have been successfully put through by monitoring specific infections and making sure that there are protective ways in place.
Mm-hmm.
I think this is like you say, it's a good question, but it's not an unmanageable one.
Mm-hmm.
Eliana Merle, UBS. Just in terms of thinking about the initial timing for getting some of this data, just given the fact that there are serum biomarkers, could we potentially see some of this healthy volunteer data, such as the single ascending dose from the RAGE program perhaps this year? Or just any color on the timing to clinical data disclosures, albeit in healthy volunteers, just even for the readouts in terms of the delivery and the platform there. As well as, could you update us on the timing of the COVID programs? Just another, you know, interesting program, as well as thinking about readouts in terms of delivery. Then I have a follow-up question as well.
Sure. The short answer is, yes, you could see data this year, but no, I can't give you guidance on when that'll come. Yeah, I think we'll start dosing healthy volunteers in the next couple of weeks. But until we start dosing healthy volunteers and the patients, and until we see how that recruitment's going, it's hard for us to give guidance. But I think your sense is right. You know, our hope is that since there are circulating biomarkers, that we could have data on the early side, and certainly this year is a possibility. On COVID, I tell you know, our data continue to be promising. Our choke point here is experimental models.
You know, they're difficult to come by these days, and so that has slowed us down, unfortunately. We're still excited about what we're seeing, and my hope is that we'll have some more data later this year that we could talk about and see where we are with respect to potentially nominating a candidate. We also didn't talk about other respiratory viruses, but as you can imagine, we are deep into others as well. We're not prepared to talk about those yet because we don't have guidance on timing yet. Those are, you know, a half a step or so behind where we are with MMP7.
Great. Thanks so much.
Sure.
Then just a question on the macrophage overload and the safety that you discussed and some of the learnings from the non-human primate data as well as some of the rat studies. Given these would potentially be chronic dosing, can you help us understand both the dose as well as the time relationship to some of these inflammatory effects being seen and your confidence in terms of longer-term dosing, even at, say, you know, much more potent chemistry or lower dose levels and longer intervals between dosing, that this wouldn't be something that happens over time? Thanks.
Yep. Thanks. James, you wanna take it?
Sure. Yeah. I think, and we'll see once we get to chronic tox studies, but I think, based on, you know, what we've seen in the acute tox study and what we saw in the ENaC chronic tox study, where we were eventually in the rat able to achieve a NOAEL at a single dose every two weeks using the mid dose for ENaC, we can spread that out quite a bit for both of the candidates, like for RAGE and for MUC5AC. I think as long as we can stay below that threshold, then the normal clearance mechanisms can do their thing to the extent that they need to, that we should be okay in chronic tox.
Yeah. I think there are two really powerful slides that speak to that. Well, at least two. You know, one is that one slide where James, you know, showed the various combined doses of ENaC, superimposed on top of where that threshold is. It really sort of met expectations, right? Where those we had combined exposure that was higher than threshold, we saw this overloading for that group where we're dosing just once a day, you know, for one time every two weeks, we are underneath that threshold, and we had NOAEL. That was compelling. Then compare that to what we expect for the chronic dosing for MUC and for RAGE, we're substantially below, looks like that anticipated threshold.
The modeling looks pretty good. We're excited about the second slide that kind of speaks to that James also showed was the two plots of activity over time with ENaC versus RAGE. ENaC, you know, came back after whatever a few weeks, whereas RAGE just clamped down. That gives us the ability to dose much less frequently. Certainly, we think monthly, but maybe every three months. We'll see.
Hi. Joel Beatty from Baird. Thanks for the presentation. For RAGE, could you help reconcile the importance of dosing locally, while also being able to look systemically at serum RAGE as a relevant biomarker?
James.
Sure. All of the circulating sRAGE is coming from the lung. RAGE can be produced in areas outside of the lung in the setting of local inflammation. In the healthy volunteer and in the asthmatic patients, first of all, when we administer via inhalation, we're only getting knockdown in the lung. Essentially all of that sRAGE in the blood is coming from the lung. There are no other significant extrahepatic sources.
Great.
Up to you, Colin.
For MUC5AC, what's the ability of the drug to make it through the mucus that will presumably be quite prevalent in these patients?
Sure. I think I have a slide that sort of addresses the overall physicochemical properties of the TRiM conjugates. The MUC5AC drug is no different than the stereotypical one we're showing. Very small size, net negative charge, soluble. When we go into models of mucus obstruction, we can do a neutrophil elastase model, for example, where we invoke significant mucus secretion. We can dose our drug and still see pharmacodynamic response and knockdown. We're confident that delivering to a higher mucus environment, we can still get mucus penetration and knockdown.
Thank you.
All right. Just one more question.
Hi. Thank you for taking our questions. This is Farzin from Jefferies. Is the macrophage overload an issue when administering subq, or is also the same thing on? Then do you see a differences in the overload based on the targeting ligand or just it's the amount on the RNAi used? Maybe it's for James or Erik.
James.
The question is the macrophage overload relevant to subq or? Right. That, yeah, there still is some macrophage uptake of the drug in the lungs and in other tissues when administered subq. It's really more of an issue when it's administered via inhalation. Though that's really when it's the easiest just given the high concentrations to overload the macrophages.
Got it. For the KOLs, one big picture question, like you're targeting upstream, so are there any obvious risks for infection further down the line when you knock down RAGE?
Could you repeat the question?
Like if you're targeting very upstream with the RAGE, and then are there any obvious risks downstream?
Mm.
Like for opportunistic infections later on?
The answer is we don't know. I mean, we know that at least in the animals that Erik presented for a year, there was no specific increase. But these were animals, and they were in a laboratory, not kept germ-free, but not that. So we're not certain. There are, you know, that was discussed, mouse knockout experiments with pneumonia, for instance, that didn't seem to influence the outcome. Actually, if anything, in a beneficial. There might be other infections. So I think it's. Is there a potential risk? Yes, but that's true for any biologic that you know, use. TNF-alpha inhibitors classically, right, they have similar risks. You just need to understand them and then deal with them. Yeah.
Okay. Well, thank you all for coming.