Stavros Zanos, MD, PhD - Neuromodulation of neuro-immune circuits

Thank you very much. Thank you for inviting me to speak at this conference. I'm really excited and happy to be here. It's one of the few conferences that I know almost nothing about the main field, but I feel very comfortable speaking to all of you. And the reason is discussions I had with Dr. Setskin before who really post the idea of what if their nerves are really involved in the pathogenesis of the disease, not just explaining the symptoms, but driving the pathogenesis. So that really caught my attention. And then I sat through some of these talks earlier and I realized that there is actually a lot of mature ideas linking the nervous system and the immune system with endometriosis. And I think this is a very valuable discussion to have. Now, I'm not going to present any preclinical or clinical data in endometriosis, just to be clear.

I'm going to discuss how we think about modulating these circuits that Ibrahim talked about for therapeutic purposes. So then maybe we can begin a discussion of whether this makes sense or some of these make sense to be applied into patients with endometriosis. The idea behind this whole neuromodulation by electronic medicine is that now that we know that there are all of these nerve immune interactions through characterized anatomically and physiologically characterized pathways, what if we tap into those pathways? What if we knowing that this nerve, when activated in the animals, it has this specific effect in the immune system. What if we activate that nerve with devices? And that's the whole idea of neuromodulation. And traditionally, it has been applied to treating neurological and psychiatric disease. We've got electronic medicine and the work we've been doing at Feinstein for the past, I don't know, 20 years, and I've been with them for nine years, is to see if we can use the same concept of neurostimulation, of engaging these neurological pathways this time to treat neurological disease.

Ibrahim talked about cardiovascular diseases. Other people are looking into autoimmune, rheumatological, gastrointestinal diseases. So that's the whole idea of bioelectronic medicine. And I want to give you a general overview of what approaches we use, what are some preliminary results from exploring these approaches. These are my disclosures. So again, this is the idea of stimulating with neuromodulation, renewal pathways that connect the nervous system and the immune system. And one major part of the immune system and the nervous system that we care about is how the autonomic nervous system influences immune function in the spleen and in other organs, but primarily the spleen.

So I'm not going to spend more than 10 seconds on this. This just reforces the idea that these pathways are reflexes. You have a sensory arm with green on the right side. It goes through the spinal cord and peripheral nerves like the vagus nerve into the brain. And from there, it's all integrated. And then the descending path or the motor path where it goes again through the spinal cord or the brainstem through spinal nerves and splacnic nerves or the vacuus nerve into a layer of organs, including the spleen. Now I'm mainly showing the spleen because it's kind of an important organ, this neuroimmune communication, but motor vegal fibers and sympathetic fibers go to a lot of other organs, including the abdomen and the peritoneum. And the end result of all of this reflex is to regulate inflammation and the immune response. If it goes crazy high, you want to silence it down.

If it's not enough, you might want to boost it. So there are bidirectional interactions, both more, sometimes pro- inflammatory, sometimes anti-inflammatory. Obviously for diseases with an inflammatory component, we want to suppress inflammation. So that's our therapeutic goal. So we can stimulate sensory. Again, this is a reflex. So we can stimulate sensory fibers and I will talk a little bit about that. We can stimulate the spinal cord. We can stimulate the motor fibers of the vagus nerve, or we can stimulate the spleen. Ibrahim mentioned the focused ultrasound of the spleen. We actually have tried it in humans, and I will show you what happens when we do that. So in terms of devices and how we interact with these pathways, we have several options. One is to stimulate the cutaneous sufferance. It happens that these cutaneous sufferings in the oracle, we call it transcutaneous oregular stimulation.

They make synaptic connections in the nucleus tractual solitarius that is very close to those of the vagus nerve. And actually the uricular nerve is considered the branch of the vagus nerve. It's not vagus nerve stimulation, but it's something that resembles it in some ways. So that's an option. It's easy. It's non-invasive. You can place a tense device there and perhaps get some effects. Then we have the traditional chronically implanted vagus nerve stimulator in the cervical vagus nerve. We can stimulate the splenic nerve directly. Ibrahim's from some data and preclinical data on that. There is a company that pursues that therapeutically, or you can stimulate the end organs. In this case, the spleen with focused ultrasound stimulation, which is what we do around with VNS, we studied that in the lab. All right. So bipolar stimulate the cervical vagus nerve by an implant. And this has been around for decades.

It's not new. Treatments like depression and epilepsy and more recently stroke have been shown to work really well in patients, but they didn't really target the neuroimmune pathways. So the idea behind that was explored by an implant from a company called SetPoint that was co-founded at Feinstein. And recently, at the end of the last year, they published a study, which was a randomized controlled trial, which is the best level of evidence we have for any new therapeutic approach where they gave people with rheumatoid arthritis a stimulator or a stimulator, which they never turned on. That was their placebo or some condition. And what they found, so the SAM is the black trace. So interestingly, they saw an effect. You can see it's like a 20% rate of responders based on some clinical criteria and laboratory criteria. That was in three months. And this is a known effect.

I'm happy to have these discussions with you, known effect with any device. You get a very strong placebo effect. First, you want to saw that the device does more than just placebo. So thankfully that did happen because at three months, the treatment group had a higher percent of responders than the placebo group, and that percentage actually kept rising. At 12 months, it was close to 50%. And again, that's a known effect in neuromodulation for pain, for depression, for other diseases that the longer you subject those patients to stimulation, the better the results are. And this time course is actually very convincing because of previous neuromodulation trials with spinal cord stimulation, with brain stimulation. That's what we expected to see. That's what these investigators saw. This is, by the way, rheumatoid arthritis that is very difficult to treat. Actually, these people were almost impossible to treat because they received a combination of anti-inflammatory drugs, biologics, and they still could not see an improvement.

So there's already a selection of patients that were very hard to treat, and they got almost a 50% responder rate, which is clinically very significant. Now, what we're doing in the lab, and I'm going to keep it very short, is we try to make better stimulators. We've been talking with Dr. Seshkin, maybe trying some of these stimulators in people with endometriosis, but the problem is with ... Well, not the problem. I guess one limitation of these devices is that stimulate the entire nerve. So you have, you remember, sensory and motor fibers going into the vagus nerve, and also you have fibers from different organs. It's not just the spleen. We kind of focus on the spleen, but there is much more happening in the vagus nerve on the spleen. You have fibers to the airways, the lungs, the heart, the liver, other organs in the abdomen, but we do want to only stimulate the spleen.

So we need better devices. So in the lab, we've been working on this for the past several years. And the way we approached it, I'm going to keep it very short, is by taking from cadavers, the vagus nerves, going deep into the structure of these nerves by doing microanatomical studies, and then finding the sensory and motor fibers in the vagus nerve, visualizing them, reconstructing them in 3D and seeing if they're separated. Are they all inter-leaved or are they separate inside the nerve? It turns out that they are somewhat separated at different levels. It's a complicated story, but to some extent, they remain separate, which means if you use a more smart device that would separate the stimulation, the side of the motor fibers versus the side of the sensory fibers, you can get a more selective VNS device. And that's exactly what we've built. We've published it and now we are moving hopefully by May we have some good results from application we submitted to the NIH and they were positive about it, about translating that in humans.

So that would be a next generation VNS device. Vericular branch, very easy. You place it on the Oracle, you turn it on, you can get these devices for 20 bucks on Amazon. It's a very compelling, at least in terms of ease of use proposal. We actually tried it in, again, randomized control trials in some cardiovascular diseases. So this was the TRITAF trial where we saw a meaningful, even though there is a low variability effect at six months. So the people who received the treatment versus those that received some had less burden of this atrial fibrillation condition. And we also used it in ventricular arrhythmias.That's a different kind of cardiovascular condition that affects the ventricles of the heart. We also saw in another randomized controlled trial that there was meaningful reduction of the incidence of the disease, even after less than three months. So the effect is moderate.

I'm not going to say it's life-changing to these people, but it's interesting because we actually didn't expect to see much. It's such a small number of fibers in the Oracle. You stimulate them once a day, twice a day for a couple of weeks or a month. I mean, you wouldn't expect to see much. However, if you are careful with patient selection and with following up with these people and making sure they are receiving their treatment, there is some small but meaningful improvement. Okay. So now let's move to the spleen because you heard a lot about the spleen. The spleen is a deep organ. It's not easy to get to it surgically. So we've been thinking about the non-invasive ways of stimulating the spleen. And as Ibrahim said, one way is focused ultrasound. How does focused ultrasound works? I mean, this is the same principle as imaging.

Actually, this is an image of the abdomen and the spleen with the same machine that we use for neuromodulation. So we use the same machine for imaging. When you go to the radiologist office, you get an abdominal ultrasound. This is pretty much the principle. Now we use different parameters, of course. We use parameters tailored to neuromodulation rather than imaging, but it's the same idea. How does it work with the nerves? These raves, these ultrasonic waves, they bring these membranes into some kind of oscillation. And by oscillating at these specific frequencies, they open iron channels in their membrane. And when all iron channels open, nerves get activated or suppressed if you do it for longer period of time. So instead of electrical, speaking electrically to those nerves, we speak to them mechanically. That's the general idea. The brain advantage that is non-invasive and it's image guided.

So we wanted to see that we have all these core results in animals like Ibrahim showed. Doesn't work in humans. And so we did a pre-registered randomized control trial where we enrolled 70 healthy subjects and there was one sum group and six foos groups, different intensities, different localizations in the spleen. It's not important. What is important is that these are healthy people, so they don't have active inflammation. And we saw that the ultrasound has an anti-inflammatory effect in humans. And I will tell you how, but the main idea is we measure TNF at baseline and you see that some and ultrasound groups are not different. One hour after the ultrasound, the insonified group is lower, two hours is even lower, and that 24 hours returns to baseline. So we get a distinct, significant inflammatory effect. And why is this significant? This is lugarithmic scale. This is a fourfold change in the levels of TNF.

This is not like a little bed you have to do statistics together. This is a significant difference. Now through major TNF in a subject that has no inflammation, we have this ex vivo challenge. We take blood from these people, and because they don't have inflammation, we need to excite them. We trigger them, trigger inflammation with an endotoxin. So we do that outside of the body. So if the people who received ultrasound, their white blood cells are less responsive to these inflammatory stimulus, that means that inflammatory stimulus had an anti-inflammatory effect.

There was no evidence of toxicities. Very safe. They didn't report any weird feelings, any perception of the ultrasound. That's why it's a real controlled study because they had no idea whether they were receiving the treatment or not. So I thought it was very cool. And we're already discussing with clinical groups. Alexandra is here. If you want to know more about the involvement with clinicians on the level of cardiovascular disease, she's working on that. So we think it's definitely a technology keep that is worth thinking about. And finally, I want to talk a little bit about the spinal cord because I heard people speaking about it earlier, and I like this idea a lot. We wanted to test the focused ultrasound of the spinal cord. Again, we started with a preclinical model, ERAD, improves pain. Well, pain, warfare goes through the spinal cord, dorsal ganglia ascending pathways into the brain.

And so we're going to test if this spinal cord neuromodulation that works in the spleen also might work to reduce pain in an animal model of pain, of chronic neuropathic pain, which is what some of you experts in endometriosis you think might be happening in some of these patients. So this is the animal that is receiving ultrasound on the back. That's what it looks like. You see that red area which extends into the spinal cord. So this is the spinal cord, this is the vertebra. This is the spinal cord. So you see it gets a little bit of energy into the spinal cord through the bone. This is not just in rats. So we know now from a different group that this also works in humans. They can demonstratively show that focused ultrasound through the bone reaches the spinal cord. So it's doable in humans.

And then we just did this treatment, three, five minute treatments after the chronic neuropathic pain was induced. So it was an actual injury to the nerve. The animals develop pain. What happens if we treat those animals with just three daily sessions of focused ultrasound of the spinal cord? But what happens is that the height of these bars and the black is fuse, white is some. The height of these bars is inversely proportional to the pain these animals feel. This is the threshold, how much stimulus we need to deliver in order to report pain. So that's the normal, that's the normal level. So they only report pain at 40 units of pressure, let's say. Immediately after the injury, that threshold drops to 15, which means they're a lot more sensitive pain systemization. And then what you see after the treatment happened here, the difference between the SAM white and false animals is there, and then it's maintained.

Now we haven't repeated the treatment. We only delivered treatment for three days. So whatever happened there in the spinal cord of these animals, in this sensory pathways, was maintained for up to three weeks.

We can actually measure that. We have these methods of measuring the activity of sensory fibers, A, delta, and C fibers. Those are nociceptive fibers that convey pain information to the brain. The animals that received ultrasound had much less activity of these pain fibers than those that did not. And finally, what we found is the spinal cords of these animals were different. So the levels of microglial activation, the numbers of microglia, which are these inflammatory cells inside the spinal cord that we know get multiplied after a spinal cord injury or a nerve injury, were less in the animals that received ultrasound than those that received the SAM treatment.

So how is that all relevant to endometriosis? Well, the short answer is I don't know. I'm not an expert in endometriosis. I'm here to learn more about it, to speak to all of the scientists and surgeons, and more importantly, I think patients. But I think perhaps neuromodulation has some role, at least consider in endometriosis, because first of all, it's based on strong scientific principles. It's not just some kind of lifestyle, somebody thought of it and they put it on TikTok or Instagram and all of a sudden it becomes a meme. This has 30, 40 years of scientific and clinical history behind it. It's also practical. You don't have to take new drugs. It's a device, sometimes it could be even non-invasive or invasive. Of course, the implanted VNS device, and we had long conversation with Dr. Setskin on that, would be the holy grail. You take a device, you forget about it, this does its job, you continue your life, but it's not easy to perform an RCT in a very new indication.

RCT means half of the people will get a device, but it will never be stimulated. How do you convince these people that it's okay to spend a considerable amount of money, even if there was a sponsor? How do you convince them to undergo this surgery without getting the treatment? So it is a little bit of a threshold to enter that. I think personally that Splen-focused ultrasound may be an option. If not for chronic treatment, at least there's an option to select patients that would be highly likely to be held by VNS. So you see if this anti-inflammatory circuit is active in these people by stimulating them with ultrasound, you take these results back in 24 hours, 48 hours. It's noninvasive, it's painless. And if they do respond to this treatment, based on what we know from the biology of these pathways, is that they are good candidates for chronic VNS, and maybe we should consider implanting a chronic stimulator in those people.

Now, it could be that at home foos could be a long-term solution as well. We don't have that device yet. We have device for short-term splenfus. We don't have a device for taking this, let's say putting a belt on, going home and programming it to stimulate your spleen on a daily basis, but I know people are working on it. So acknowledgements, this is my group.

Our collaborators and funding comes from both federal and industry partners. Thank you so much. I'm sorry I went three minutes over time.