Dr. Daniel S. Kohane - Drug Delivery Systems for Pain, Opioid Abuse, and the Treatment of Vascular Malformations

Hey, good morning, everyone. Uh, welcome to Grand Rounds. Um, today, I have the honor of introducing Doctor Dan Kahane. Uh, Doctor Kahani obtained his MD and PhD in physiology from Boston University. He subsequently competed completed a number of different, uh, training programs. The first was, uh, pediatrics here at Boston Children's, and then went down the road to complete his anesthesiology training at the Mass General, and came back here for his pediatric critical care fellowship, um, and here at Children's, and then went back to uh Mass General, where he started his, uh, faculty position, and then We ultimately recruited him back here in 2007, where he has been with us since. Uh, he clinically practices as a, um, attending in the, uh, MSICU. Uh, up there, he's received many accolades for his teaching among the trainees. Um, a, a big part of his role here at Boston Children's is, uh, in his research. He's the vice chair of research in the Department of Anesthesiology and critical care. Uh, he's, he directs the impressive laboratory for biomaterials and drug delivery, um, that focuses on, as the name suggests, biomaterials, drug delivery, and nano medicine. Um, so, a lot of very cool things. I was reading through his various projects, and, um, I don't wanna, um, Give any uh hints or spoilers about today, or what he's gonna talk about, but some of the things that that are working on there that I thought was very cool are some novel long-acting anesthetics, uh, drug delivery systems that can be triggered to deliver drugs by different stimuli like light and pH, as well as, um, targeting drugs directly to different areas in the body, such as the eardrum and, and the eyes. So all that stuff was very, was really fascinating, the work that he has going on there. Uh, he's been very successful. He's been NAH funded for many years, uh, over 200 publications, and, uh, 20 patents. Uh, perhaps most impressively though, when you review his his CV is the number of people that he's influenced as a mentor. Uh, he had 6 pages of his CV full of mentor of mentees and trainees. I'm not even sure I know 6 pages' worth of people, but I could simply write down on a page. So it's really clearly he's had a huge influence. Uh, and the many people who have been through his lab and learned from him. Uh, Doctor Kahani, you're doing such amazing work. We really look forward to hearing today. Thank you for coming to speak with us. Um, thank you for that introduction. I'd also like to thank whoever. Need to be thanked for the fact that I'm here talking to you today. Uh, I also want to apologize, um, that in an effort to, um, make everybody happy and also to, uh, highlight, um, all the many years of collaboration between my lab and your department, um, this talk is a little bit more diffuse than it would normally be. Um, so, I'm gonna talk about drug delivery systems for a whole bunch of different things. And, um, I'm gonna start off by talking about these two things. These are depot drug delivery systems, and the example I'm gonna use is prolonged duration local anesthesia. And then, um, Triggered drug delivery systems, and I'm gonna be speaking about on-demand local anesthesia, but also on triggerable opioid reversal systems. And basically, in those two topics, I'm gonna show how you can go from having your typical drug administration single bolus peak, to having either sustained release or some combination of sustained release and then triggered release. Uh, or just, um, triggered release, and I apologize for the quality of the, the slide, it's sort of like me, it's not beautiful, but it's effective. Um. So, and then, um, we're gonna talk about sensory selective local anesthetics, and then uh nanoparticles to treat uh uh vascular malformations. So, we'll start with the uh basic depot. And so the idea here is that you have some drug, drug delivery system shown here in green, that contains a drug, which is shown here in red, which is injected, uh, let's say into interstitial fluid by a needle, and that's the, the, the interstitial fluid here shown in blue, and then the drug diffuses out, and depending on the application and the drug could either be for systemic use, so the drug goes throughout the body, like. With subcu epile, let's say, uh, or it affects the pink thing over on the side, which is some tissue, um, as is the case with local anesthetics. And so, you all know about local anesthetics, they uh work on peripheral nerves. Ideally, and they're very effective actually, but the one of the main problems with them is they just don't last very long. So, there have been decades of research of people trying to use drug delivery systems to extend the duration of effect of uh local anesthetics, and I've actually been doing this since 1996. And there Many of the uses, main uses that are proposed are here. So, one is, so you can have prolonged local anesthesia throughout the post-operative period, maybe so the patients are comfortable, maybe there's a physiological benefit like after a thoracotomy. And also, maybe it would be nice if people with cancer pain or other forms of chronic pain could have prolonged pain relief. And these were the main reasons when I was starting off to do it. But for the last 10 years or so, there's also been the fact, the idea that if you could have prolonged duration local anesthesia, you wouldn't have to prescribe opioids to people, and maybe you could mitigate the opioid epidemic. And so, just to go over what the design characteristics were, um, the idea is to create something that's initiated by a single injection. It's easy to administer. You don't have to go to the OR, uh, or have general anesthesia to initiate this thing. And importantly, from that single injection, you get days to weeks of local anesthesia, and There it shouldn't do anything bad, like you shouldn't have horrible inflammation, there shouldn't be neurotoxicity, it shouldn't destroy muscle locally, it shouldn't be systemically toxic, um, particularly if you might need to be injected again at the same site, it should be fully biodegradable, and also it should eventually wear off. And so, we started working on this in 1996, and we did a whole bunch of things which did not work for one reason or another, or it worked, but there was um an untoward side effect. So, for example, all the sustained release systems that use conventional local anesthetics like buppivacaine or whatever, would give you long blocks, but the tissue reaction from the drug was really bad. Um. And so, eventually, um, after about 13 years, we came to this formulation, which is liposomal sax toxin. So, Liposomes are basically particles that are made out of lipid bilayers that contain drugs, and you see a picture of one there. And we used um saxitoxin, which is a site one sodium channel blocker. And uh You can see on the left two examples of site1 sodium channel blockers as tetratoxin or TTX, which is what kills you when you eat fugu sushi that is not prepared properly, and there's the saxitoxins that, uh, which is what kills you when you get paralytic shellfish poisoning. But the good news is that if you can prevent them from going systemic, uh, if you keep, keep them localized, they're actually ultra potent local anesthetics. And so the way they kill you is the same way that they provide local anesthesia. They block sodium channels, and so when you inject them, being very hydrophilic, they basically go swimming throughout the body looking for sodium channels to block, and they pass the phrenic nerve, and they say, oh, I'll block that, and then you stop breathing and you die, and they do something similar at your sympathetic nerves and you become hypertensive, but importantly, Uh, they don't cause arrhythmias, they don't cause seizures, and they don't cause local tissue injury, which is, uh, all of which are features of conventional local anesthetics. So, again, the concept is if you could keep it localized at the nerve, you would get very powerful local anesthesia without all the baggage. And so, these data show you what happens if you inject these liposomes of saxitoxin at the um sciatic nerve in rats. Uh, it's nice actually for once to talk to people who know what the sciatic nerve is, um, cause I usually talk to chemists and stuff, but anyways. So, I want to draw your attention to uh two points. One is this one, solid STX. This is actually solid liposomes, which means liposomes where the lipids have a relatively high melting point, which slows uh diffusion of the drug out. And you can see that with the solid saxitoxin liposomes from a single injection, we got two days of nerve block in a rat. But for context, um, 0.5% bupivacaine, um, or marking or sensorcaine gives you in the same model, 2.5 hours of nerve block. So, this is, uh, quite a bit longer. And if you add just a wee bit of dexamethasone, you can see that the duration of block from a single injection is 1 week. Um, the idea of adding dexamethasone to local anesthetics was actually, uh, done by Chuck Birdy and Robert Langer, who's at MIT, uh, 10 years previously. And In these animals, there was no systemic toxicity, uh, but the, the real issue, uh, was how about biocompatibility. And so, Um, for those of you who've dissected rats or who have prepared, um, chicken in the kitchen, you can see that this tissue actually looks pretty well. You can see the particles sitting right on the sciaticic nerve, and the tissues around it look good. And to make a long story short, histologically, there's a little bit of inflammation because anytime you inject anything, including saline, into the nerve, um, yeah, sorry, into the body, you get some inflammation. Uh, but also, uh, but there was no, um, actual tissue damage, um, unless there was bupivacaine in the mix, and high resolution microscopy of nerve showed that the nerves were intact. So that was in 2009, and uh over the years that followed, we needed many things um to try to make this even better because in order to prolong the effect further, you have to increase the dose of TTX and eventually, you'll get toxicity. So, the, the, the idea is to make systems that make release slower and slower and slower. And so, you can see here, um, 14 years later, there's this thing by Yang Lee, uh, in my group, who, uh, this was published in Nature Communications. And here, the idea was to modify the lipids that were in the bilayer so that there were aromatic groups at the end of the acyl chains, so that they would interact with each other on the inner leaflet of the lipid bio. And we showed that this approach would slow the release of drugs from the liposomes. And um these are the in vivo data um uh after sciatic nerve blocks with TTX containing liposomes of various types. And so in both graphs that you see here, the X axis is the dose of TTX that was injected. And in the top graph, we look at, uh, the Y axis is the duration of block. And what you can see is that with conventional liposomes, um, the dose of TTX increases. And when you get to about 20 mcg per rat, which is 4 or 5 times the lethal dose for free TTX, you get a duration of a block of about 20 hours. And then when you go a little bit higher, they actually start dying. And then when you get close to 30 mcg, they're all dead. Whereas if you look at the blue line, um, As the concentration goes up, you can go all the way up to about 50 mcg per animal, and that, and at that dose, you're getting 3 days of nerve block from single injection, and there is no mortality. Um, and if you just add a teeny amount of dexamethasone, you get 8 days of nerve block. And you may say, well, that doesn't look all that much better than what was done previously, um, what we did in 2009. But TTX is actually one half the potency of saxitoxin. So this is actually a pretty marked improvement. The other thing is that, um, One could have increased the loading further. Uh, this was last year, but since then, we've actually found something much better, which we'll get to in a moment. So, we sort of lost interest and moved on. Um, so, another, um, So, we have looked at many different ways of trying to achieve a prolonged uh nerve blockade, not just liposomes, all kinds of particles and binding drugs covalent to polymers. But one approach that was very original actually, that was um done by Dali Wong and someone you may know, um, Chris Weldon, was to use aptamers as drug delivery systems. And so aptamers, as you may know, are single chain, uh, uh nucleic acids, um, And you can select them by a process called sex, that so that you find um sequences that bind. A drug with relatively high affinity. So, I think it's sort of like an antibody, but instead of being a protein, it's a string of nucleic acids. And so, Aptamers have been used quite a bit as, um, as for example, you would bind a drug to an aptamer, and the aptamer is specific to some antigen in the body, and then you inject it intravenously, and the aptamer carries the drug to um a specific target in the body. But the idea that Dali and Chris came up with is to use the aptamer itself as a drug delivery system. And so, what you would do is you would mix the antibody with the aptamer that it's specific to. And then inject those two together at the sciatic nerve. And because the ner binds TTX, it will therefore release it slowly. And to make a long story short, that worked, and this system could provide prolonged duration, uh, local anesthesia. Um, so, this is the latest thing, and I apologize that I can't be detailed cause it's neither published nor patented, but, um, this is a liposomal system that is releasing TTX. And this is um the data from a single injection at the sciatic nerve. So on the X-axis, this time after the injection in days, and the Y axis is hot plate latency. So you, after injection, you put the rat's hind paw on a hot plate, and you measure how long it takes for it to lift its foot off. So, 2 seconds is baseline, 12 seconds is maximum, after which we remove the hind paw. So, 12 seconds is complete block. And so what you can see is that in the absence of any adjuvant drugs, there's 13 days of nerve block, and here, nerve block means time to return to the halfway point between 12 seconds and 2 seconds. So that's almost 2 weeks of nerve block without adjuvant drugs, and it's 21 days to complete resolution, and there's no local or systemic uh toxicity. And if we do add a little bit of adjuvantic drugs, right now, uh, we haven't found the maximum, but from a single injection is 40 days of nerve block without any apparent local or systemic toxicity. And the thing that's exciting about that, that now we're really talking about maybe being able to treat, uh, chronic pain conditions. So, we're pretty excited about this, but, you know, so what's not to like? So, Here, here, there's actually two things not like. One of them is this. What, let's say you get one of these, and in a human it probably lasts longer than a rat for somewhat complicated reasons. And let's say that on day 4, you decide, I don't want this anymore. Well, that's too bad, right? You know, you, you'd have to like convince a surgeon to like maybe open up your shoulder or your butt and take, scoop the stuff out or something. There's no real way to get rid of it. So, um, this introduces the concept of triggered nerve blockade and um. To, uh, it's actually illustrative to discuss how the idea came about. So, um, when I was a young man, um, I, um, had a big cavity, and my dentist, by my dentist, this is in Switzerland, did not believe in local anesthetics. Um, so he did everything just bo natural, as they say, and so you better believe I, I can take pain. So, um, so he drilled it down and Put a huge filling in. And about 40 years later, I guess it started getting old, and, as did I actually. And so, I was able to convince him, my, my dentist, different dentist, this guy believed in local anesthetics, to um deal with my old filling. So he injected me, I guess, at the superior alveolar nerve, numbed up half my face. And then he took the filling down, and he saw that there was this big cavity and a crack was running down the bottom of it. So he took out its drill, shaved the um crack away, and put in an even bigger filling in the bottom. And uh as I was leaving in that wonderful euphemistic way that dentists have, he told me, you may have a little sensitivity later tonight. So, later that night was actually my wedding anniversary, and my wife and I were out at a restaurant, and as we were leaving, suddenly, I felt like someone had rammed a spear through the roof of my mouth. And what that was, was the local anesthetic wearing off. And I remember thinking to myself, wouldn't it be nice if when my dentist had numbed me up, he had injected me with something that I could turn back on. Um. By the way, uh, just to finish the anecdote, so what actually happened is, you know, I suffered through the night taking Motrin, which actually didn't work all that well. I went to see my dentist, and he said, well, you need to see an endodontist. Um, but the good news is, here's a prescription for some opioids. So I went to the pharmacy, got myself a jar of some narcotic, and I took one, and I actually did not like what it did to me. But you can imagine that in an alternate universe, I might have really liked what it did to me and become addicted, or someone could have stolen it, and um either sold it, or overdosed on it. And so, a lot of this is about finding ways to avoid. Opioids. And so, the concept is to have something that is initiated by a single injection. It's not a systemic treatment. It's not opioid. You would be able to act over an extended period, but the patient would be able to determine when they get pain relief, how intense that pain relief is, and how long that pain relief lasts. And we've done many different ways of doing this. I'm just gonna show a couple. And so this is a liposomal formulation made of lipids, which have a lot of double bonds in them. And also inside the lipid bilayer is a photosensitizer, and the active payload is a mixture of tetrodotoxin and dexametatomidine. And the concept is you'd inject these things near the sciatic nerve, and then you'd shine near infrared light onto um where you had injected them. Near infrared light can penetrate quite deeply into tissue. And it would make the photosensitizer kick out reactive oxygen species, which would peroxidate the double bonds in the phospholipids, which would transiently poke holes in the membranes, and that would allow the drug to escape, which would result in nerve block. And so this is an example of the in vivo data, and I'd like to draw your attention to the red line here. So you see that you inject, uh, so that the X axis is time after injection, the Y axis is a metric of local anesthesia. So you can see with the red line, you inject at time 0, and you get this nerve block that lasts for about a day and a half. And then when it wears off, uh, a day and a half later, if you shine a light. Near the greater trochanter, where the particles are sitting on the nerve, uh, when you shine the neo red light on it, you can see that the nerve block comes back, and then it wears off, and you could do it again and again and again. Um, in theory, if, if the rat had free will in this, it could do it when it wanted to. And in this particular case, we could get 9 decrementing additional local anesthetic peaks, which in this case added another day to the duration of effect. Um, I like, there's 3 examples. So by swapping out the photosynthesizer for for a sonar sensitizer, um, which releases reactive oxygen species in response to ultrasound, uh, we are able to do essentially the same thing, but using ultrasound, and there's pros and cons to using ultrasound to, to, uh, trigger nerve block. Um. But there are some problems with these um systems. So, first of all, as you saw in the, one of the, the most recent example in vivo that I showed you, you get this 1.5 nerve block. What if you don't want to have a 1.5 nerve block for starters? And the, the reason you get it is that these liposomes rely on diffusion for the drug to get out. So there's this basal release, which you can't turn off. Um. And the other thing is, well, you cite one sodium channel blockers, they work extremely well, but they, for most of the world, they're relatively um exotic or perhaps scary compounds. So, for translational reasons, it might not be nice to use more conventional compounds. And so, Uh, Wei Zhang in my group, um, came up with, uh, this schema. So it's, um, two molecules of tetracaine, which you are all familiar with, which are bound on either end of a polymer by a photo cleavable linkage. And so, the idea is that after this thing is injected, if you shine light on it, It will cleave right at the blue pink interface, releasing tetracaine in its native form, and the polymer is actually an interesting polymer. has reverse thermalmodulation, which means that as you heat it up, it actually gels, which is the opposite of most materials that we know. So, in a syringe, it's a liquid, and you inject it into a warm body, and it forms a hydrogel, so it stays in place. And these are the data in vivo, so it's injected. And the X-axis is time after injection. And there's two important points that are in the red circles. The first is after injection, you see, there is no nerve block initially because the tetracaine is bound covalently to a molecule, uh, inactivating it, uh, until you shine the light and you can see where the blue arrows are, you shine the light. The drug is released and you get nerve block, and you could do this again and again. And my lab is actually still working on the again and again part cause we'd like it to be like for several days. Um, so, in all the examples I showed you, it was triggered release or local release, uh, or untriggered release for local use. You inject it and you're trying to affect the tissue right next to it. You can also do this for systemic drug delivery, and this is an example of this. So, here, it's the same concept. You have a polymer on either end, you um tack on naloxone or Narcan, and then you make nanoparticles out of it and you inject them subcutaneous subcutaneously. And what we showed is that um when we, we, we could inject these things into mice subcutaneously and they're well-tolerated, and then uh we would give the mice a large dose of opioids. And then when they were blissed out in that way, if we shine light of a certain wavelength on the nanoparticle depot, it would release naloxone, and it would um sort of reverse the effect of the opioid, and the, the idea is that for people who are at high risk uh of uh opioid overdose. You know, their, uh, healthcare practitioner would give them a subcutaneous injection of this stuff, and maybe also give them a medic alert bracelet or some other adornment that has a blue light in it. And so, when they feel themselves fading or their friends see that they are fading, they would just press a button on the adornment, and it would shine a blue light on it, and uh they would be uh resurrected. So, getting back to this. Um, latest, uh, nerve block thingy. So, the other thing that one might not like about this is that local anesthetics don't just give you analgesia, they also give you motor block. So with this, you are also going to have motor block for as long as you have sensory block, and you can imagine how It may be better than being in excruciating pain, but it's still kind of a drag. So, that brings us to the topic of the sensory selective local anesthetics, and um. This is work that was spearheaded by Claire O Ostertaghill, who many of you may have known as a vascular malformation fellow who was here for about 3 years. And what I can show you is a small fraction of the work that she did in my lab, which she did on top of all the clinical papers that she wrote with Doctor Dickey and all the work that she did actually being a clinician here, um, she's now, um, back doing her surgical residency. So, um, this is about a molecule called DPX. The full name is written below. And actually, this is a story about how important controls are. So, we were looking at the differential effects of various local anesthetics on nerve block, and so, we explored. Amino uh amide local anesthetics of this, with this particular nucleus. And you can see they all are very similar, except where that red circle is. So, on the nitrogen, uh, on, in the piperidine ring, The difference between all those local anesthetics is just the number of carbons on the carbon chain. So bupvicaine is 4, repivacaine is 3, mpivicaine is 1, and I was just curious, so what does, what happens if there's no carbon there? So, and that's what PPX is. And this is what it does. So, if you look at the molecule on the, if you look at the uh left panel, that's ropivocaine, a local anesthetic that uh many of you know. And I know that the literature says that in humans, there is a mild sensory predilection, but in rats, in our hands, the, the, as you can see here, the duration of sensory and motor block is exactly the same. But when we injected PPX, We found a striking. Sensory predilection. Um, and so we were pretty excited about this, um. And, you know, why, why we're excited, cause there's a bunch of contexts where having sensory selective block could be nice. So for example, in labor epidurals, it would be nice if you could make the parteron completely comfortable or reasonably, much more reasonably comfortable without them being unable to push, without getting arrested labor. In many orthopedic and other types of surgery, it would be nice if the patients could participate in the rehab, but also be pain-free. Um, and of course, in chronic pain, it would be nice if you could have, um, a prolonged period where, um, uh, the patients are comfortable, but, um, are still able to ambulate. And so, um, Claire went on to do dose response curves. And what's, the only thing that's important about this is, uh and it shows the importance of being really, you know, pharmacologically rigorous and not looking at just one dose. I think some people have looked at PPX's effect. They looked at just one dose, like down around 0.4% and saw nothing, cause it's not a very potent local anesthetic. But, what we found that there's a, there's a defined range in which it is sensory selective. If you're too low, you get nothing. If you get too high, you get non-sensory selective nerve block. Um, the other thing that was exciting to us, that we tried this intrathecally. We would have done epidally, but we didn't have the animal model. And so intrathecally, we found that ropipicane was not particularly sensory selective. Um, or it was actually be more accurate, it was not reliably sensory selective, whereas Compound X, which is actually PPX, was very reliably, uh, sensory selective. Uh, and so, to mimic a continuous effusion, we did repeat the dosing with um, Uh, PPX and found that we could do continuous sensory selective local anesthetic. And to me, this suggests that if we did have a continuous infusion, it would, uh, work. So, um, the question is safety. So, interestingly, we have not been able to find any reports in literature about the local anesthetic effect of PPX. Uh, PPX is actually a metabolite of all the other local anesthetics of this class. So people have been studying it for decades, but from its safety point of view, and it is known that as a a good safety profile. And um if you think about local anesthetic toxicity, Basically, as we showed in the paper 20 years ago, rats go through this sequence of events. They get rescued uh they, they get rescued distress, then they become numb in the extremity opposite to the extremity where you did the nerve block because of systemic distribution of drug, then they have seizures, and then they die. So, and 25 years ago, we showed that the LD50ropivacaine in adult rats is about 56. Milligrams per kilogram. We did not want to do LD 56, uh studies again, uh, sorry, LD 50 studies again. So, we just chose a dose where we knew they were gonna get sick, but they weren't gonna die. So we did 40 mg per kilogram, and then that, as you can see in a lot of those metrics of toxicity. The animals injected with the ropivacaine were quite toxic, but when we injected about on on a molar basis, actually that is twice the dose. When we injected twice the dose of PPX there was no toxicity at all. So that suggests that it's actually quite safe. And um when you looked at, when we looked at tissue reaction, um, which means inflammation, myotoxicity, and also high resolution microscopy of nerve, um, the, uh, there was no nerve damage and the inflammation and myotoxicity was comparable to what you would get with, uh, ropivacaine. Uh, this is, sorry, as I mentioned, the nerve, uh, to a neuropathologist was indistinguishable from ropivacaine or actually indistinguishable from saline. Um. And yes, so, and we have found some ways to extend this effect for some time. Um, so, basically, to summarize the PPX findings, a small molecule, sensory selective nerve block that works on peripheral nerve and intrathecally, low systemic toxicity, and a tissue reaction that's as benign, at least as that of um uh ropivacaine. And then there's this, and again, I apologize, this is neither published nor patented, so, um, I can't go into details, but this is a liposomal formulation that Yuan Han and my group developed. And so, these are liposomes with a drug combination, and um I have to admit, we don't actually understand how this works. But, but it does. And um, so what you can see, if you look at the purple line, if you do the 1 X dose, so to speak, of the formulation as it exists right now, you get about 18 days of nerve block from a single shot. The thing is, there is no motor block. It's all sensory, and we've actually shown that if you do this intrathecally, you don't get quite the same duration yet, but you do get um sensory selective nerve block, and the, the other things are sort of fractions of the 100% X, and you can see that if you give a lower dose, you may not want 18 days of nerve block. It gets um shorter. Um, finally, um, I, I, I, I, I think it's finally. Um, I'm gonna talk about, um, things that we've developed to treat venous malformations, um, and, um, this has been the product, uh, of either active collaboration with people from your department like, uh, Claire Ostertag Hill, uh, or funding from your, um, uh, the Weitzmann Fund in your department. And so, Claire participated in this, and this was spearheaded by uh Doctor Kate Cullian, who many of you know is in the uh medicine ICU uh here uh as an attending. And so, the idea actually came from a time when Kate and I were on service together. She was a fellow, I was a Um, not young, but younger attending, and um, we posited that maybe the use of nanoparticles would enhance the pharmacotherapy of venous malformations. There aren't many or any perhaps effective treatments for venous malformations, and so, Just say, so nanoparticles basically are particles where the principal dimension, whatever that means, is less than about a micron. So, it's in the in the nanometer range, but usually in drug delivery applications, we're talking about less than 250 or less than 100 or um even less depending on the context. So, this is a bunch of silica nanoparticles that uh Kate made. And um so the reason we thought that nanoparticles would perhaps work is the following phenomenon, which is called enhanced permeation and retention. So when you inject nanoparticles intravenously, they go throughout the bloodstream, and in most arteries, they just go in one end and out the other. But in some tissues like in tumors, where the vasculature is leaky, They actually extravaslate, and they don't go back in for a variety of reasons. And so what happens is you get a preferential accumulation of drug. Inside um these tissues, and we had shown Earlier that this didn't just happen in tumors, that it happened, for example, in infarcted left ventricles, it happened in choroidal neovascularization. And so Kate and I posited that there would be an EPR like effect in venous malformations also because the vasculature is not normal. And one of the things that encouraged us was this X-ray that we saw on a patient that we shared, and this was actually a patient who had a, uh, I believe a lymphatic malformation, um, and, um, The patient had had a, a KB for some reason, I can't remember, and we saw this on it, and this was dye left over from uh some contrast study they had had done like weeks or months before we met the patient. And when I looked it up, this contrast agent was a nanoparticulate. Uh, formulation, so it's nanoparticles of dye, and so we can, what we assumed is that these things had extravagated, extrapolated, and now they were stuck there. And so, Hey, um, um, reached out to Elisa Boscalo, who's, I believe in Cincinnati or she was at the time, for her model of venous malformations that is made using human cells derived from, uh, human patients with venous malformations, and in the top picture you can see what it of of the venous malformation looks in one of these mice, and in the graph on the bottom, it shows that. It stays stable for about 3 weeks and then eventually it volutes and goes away, and what the bottom right panel shows you is that the venous malmation is full of these big, boggy, baggy um blood vessels and the, the, the, the fluorescent microscopy picture shows you that actually those are human cells, not mouse. So, um, that those VMs are um from what we injected, not some endogenous thing. And so, you remember that picture of all those silica nanoparticles. So Kate asked the question, so what is the ideal particle size that we should use to encourage the accumulation of nanoparticles in the venous malformations after a systemic, uh, intravenous administration. And to make a long story short, she found that the highest accumulation happened with particles that were 20 to 50 nanometers in size. And so there's two programs going on. One, which is showing very promising results, has to do with using nanoparticles to deliver drugs to preferentially to venous malformations. But the other one is to just to try to flat out destroy them with heat. And so, another project that Kate did was to make these gold nanoshells. So, these are nanoshells that are, they're hollow, that they're made out of gold, and um, What you see in the graph on the top right is that these gold nanoshells absorb light in the near infrared range, 700 to 900 nanometers. And what that means is that when you make nanoparticles of a certain geometry and size out of noble metals, and you shine light of a certain Near infrared wavelength, they heat up through a surface called surface plasma and resonance, and what the graph on the bottom shows is that when you shine light, the more light you shine, the hotter these things get. And so, He grew these. Venous malformations in animals and then injected them intravenously with gold nanoshells, and what this graph shows you is that saline or light shone on the venous malformation or um had no effect on the size of the uh venous malformations. Gold nanoshells, if injected without a radiation of the venous malformations, had no effect on the size of the venous malformations. But if you injected them with venous malform uh with a gold nanoshells, and then you shone near infrared light on the um actual VM, you can see that the size decremented and stayed down. And on necropsy, this is what you see. You can see that in all the other groups, there's these big malformations, they are, you can see from the color that they're filled with blood, whereas in the ones injected with nanoshells that were also irradiated, in two cases, we couldn't find anything, and in the others, um, there are these little nubbins left, which were not particularly blood colored, and um that. Impression from color is reflected in histology. You can see, for example, in the animals that just got nanoshells, you still have these big, useless blood vessels that stained positive for a human endothelial marker, whereas the ones that were also irradiated, basically, everything was destroyed. There were no blood vessels, basically, and almost no CD31 staining. Um, so, this basically shows the good news and the bad news. The good news is all the way on the, um, right, which is that on necropsy. Basically, with this, this, this therapy, the VMs were ablated. The bad news is that in all these animals, there were, there were, um, at least some degrees of burns, which would, um, uh, scar over. There are ways to work around that and actually it would not happen at all with um VMs that are deep, uh, inside the tissue. But in any event, we're working towards engineering that away. Um, so, in closing, I would just like to acknowledge, um, all the people in my lab, cause I've only named a few people, but, you know, it takes an army to get this done. This is a relatively old, uh, picture of, uh, my lab. And, uh, I, uh, would like to thank the NIH for having funded me for some time now. But Also, I'd like to thank you guys, uh, Department of Surgery A for sending me people like Claire to work with me, but also, um, Kate and I have benefited a lot from the funding that you see, uh, there, which actually has supported a lot of this work. And also, I should thank my own department, which pays my salary and stuff like that, and actually supports my research, uh, to a non-trivial extent. Um, so thank you for your, uh, attention, and if you have any questions, I'll be happy to take them. Well, Dan, thanks so much for that, um, that tour. I, um, I told him before he started, I hope he, you know, I, I've seen some of this multiple times, presented by him, presented by Kate, presented by Claire, and I said, I'm gonna start to understand it if I, if, if you put it in front of me enough times, and I'm starting to understand it. Um, but I think that what we see is translational science. Um, at, at its best in an environment like this, a pediatrician, anesthesiologist, intensivist, basic scientist, the collaboration that you have demonstrated with surgeons, particularly our department, uh, mentoring and, uh, collaborating with, with Chris Weldon for many years, uh, some people don't know that, that's what Chris is doing when he's not, not, uh, uh, when you don't see him, uh, he's hanging out with, with Dan, uh, and, um. Uh, several of my own, uh, personal and, and, and, and Doctor Dickey's, uh, uh, research fellows, uh, you have made them extraordinarily successful, uh, and maybe they've helped you a little bit too. Um, but, um, you actually gave credit to your predecessors, right? You mentioned, you know, Chuck Birdie and, and, and, and Bob Langer, you know, adding steroid, you know, to, to enhance, uh, um, duration. Um, and one of the things I was thinking as you started talking about slow release, um, give a little bit of history, as I'd like to do, so in this auditorium. Next to Judah Folkman's picture. So, only a few people in the room probably know this, Tudor Folkman. Invented slow release technology. And Bob Langer, who is a mentor and a collaborator of, of Dan's, um, is an institute professor at MIT, which is way above an endowed professorship. There's only a few of them, uh, and is the most cited engineer in history. Um, his very first patent. was when he was a postdoc in Tudor Folkman's lab and was on slow release technology, and they didn't make a dime off of it. And it was given away for free, and Norlant, which some of us remember was the first implantable, um, Uh, subcutaneous, uh, birth control, uh, was developed from that patent, and to this day, um, Dan's collaborating with, with, um, with Bob, who went to found, who went on to found Moderna, etc. um, and is, and is the chair of our scientific committee of our board, um. The, the, um, Um The interplay between our departments, between our history, you showed the venous malformation model, uh, that Alisa Boscolo developed actually, although she was in Cincinnati when Kate reached out to her, she developed that model here in Joyce Bischoff's lab, uh, uh, with our tissues, uh, uh, um, Joyce Bischoff is in the vascular biology program. So, um. I just wanna add some history and, and you, and thanks for your willingness to, to collaborate with um with surgeons who wanna, wanna try and be scientists, uh, and you've successfully made them. So, uh, questions from others, comments. Thank you again, Dan. I always learned so much more. Um, you're talking about some of these products as post-operative pain management. Are they at the level where you could actually use them operatively for the operation and avoid the need for general anesthesia or whatnot? Um, so the short answer is, well, it has to be shown, but in theory, it could be used the way any local anesthetic would be used now. So, you know, uh, there are some cases that are done entirely under local or spinal or epidural. Um, there's also combinations like you, you, you, it's not uncommon to give someone a caudle or something like that, and then use a general anesthetic also. Um. I, uh, I don't think it's ever been done with something like this, uh, except there was one group in Chile that used the free drug neo-saxitoxin. Co-injected with epinephrine and buppivacaine, which gives you a block lasting about a day. And they did it pre-op, and they did the surgery and postoperative care with that. This was quite some time ago. Um, There are, it's worth mentioning that there are products, sustained release local anesthetics out there, like there's XPL, which some of you may have heard of, which is this liposomal bupivacaine thing. There's a lot of controversy in literature where it actually is better than buppivacaine. So they claim it gives you a day of nerve block. And so we have on, in 3 separate papers actually reported what it does in the same animal model that I've shown you here. And in our hands, it gives a nerve block lasting 4 to 8 hours, OK, as opposed to 40 days. So, um, the answer to your question is, in theory, all these things could be used the way all local anesthetic and local anesthetic delivery devices that are currently used. are used. You, you should be able to do it for nerve blocks pre and post-op. You should be able to do it um intrathecally and so on and so forth. But you, um, you know, infiltration block, whatever. And a very nice overview of uh quite an elegant work. At the risk of asking the obvious, has anybody ever tried to adapt the technology of insulin pumps for on-demand local anesthetic delivery? Nowadays, some of them are smaller than a matchbox, wireless, wirelessly controlled. They can control from a phone. And the maximum bolus can also be predetermined, so it can be arguably safe. And it's kind of a no-brainer. You can put a catheter where you want, even surgically perhaps, and just simply adapt it outside to a pump. Anybody thought of that? So, um, the probably most, so I know people have done implantable pumps with local anesthetics. Um, I don't know if people use, um, programmable or modifiable pumps. Um, and which does not mean it hasn't done, been done. It's just, it's not something that I know. Uh, conceptually, it, it should work just fine. I, I, it has to do with like whether, uh, you know, um, I, I don't know. But in theory it would work, um, it's just that you would have to implant it. Thank you. That was amazing. Um, one thing that sort of, it's more of a comment, but I know Tom Jackson, Craig Lilla, and others in the room who do this one particular operation frequently of chest wall surgery for pectus excavatum. It's actually a whole industry now using a device to achieve what you're achieving with, with your blocks, um, with cryoablation for these nerves and adds like an hour to the case, and, but it basically achieves. Not even as good results as what you're achieving with your blocks, and it's an operation that is perfectly suited for a long acting block because these children are in pain for, you know, a couple of weeks after surgery and, and that's why, I mean, people in Europe are actually doing this cryoablation before surgery, putting through two anesthetics to do the cryo and then do the operation. And so, uh, to Terry's question, and so I mean. When you get FDA approval, let us know. Yes. Yes, um, I look forward to that day. Uh, thank you for a wonderful lecture. Uh, just to follow up, uh, um, the question that Ferro asked, uh, you know, sometimes in surgery, uh, we do Stonehenge type things, and one of them is freezing these axons, and then we hope for the best that, uh, everything will turn out, and it seems to be OK. Uh, but I was wondering what you thought in terms of first principles of freezing an axon and whether it really does regenerate, uh, normally. So, um, There, there are reports of It's not working, or, but there's reports of everything not working, um, and, uh, development of neuropathic pain and so on and so forth, um. Actually, in talking to pain experts, one of the areas of greatest interest, particularly with in relation to the thing that lasted 40 days, was exactly the kind of context you're talking about before going and doing something that ablates a nerve, uh, which means that the function is lost forever with whatever motor and other effects it could have, to at least first try just blocking it for a month and seeing, um. If things get better. Um, well, first of all, if it doesn't get, you can do it again, but there's actually a very confusing literature that we have contributed to, and we actually did not help, um, about whether prolonged duration local anesthesia helps with, for example, crush-related neuropathic pain development. And 3 groups. Including my own, have tackled this. And so the basic paradigms, you do a crush injury on a nerve, and then, um, in the two other cases, um, you have a sustained release system that releases, let's say, a buppivacaine or a conventional local anesthetic. And in ours, we did repeated injections with um the liposome that I showed you that has saxitoxin and dexamethasone. And basically, there was the, the answers from the three different groups where it works, it doesn't work, and maybe it works. So, one group showed that their treatment completely removed the, the pain. The other one showed it had no effect whatsoever. And in our case, we showed that there is an effect, but only as long, um, the longer the block, the longer it takes for the neuropathic pain to develop. But uh, but eventually it'll happen again. So, I can't really, um, at this point answer whether um Having a prolonged block would actually fix anything. I will say this, when they were blocked, they felt no pain. Where that's worth. Dan, thank you for, for what's really been a fascinating, uh, presentation today, and, and I feel like it's a window into the future and what our, our specialty might look like, uh, uh, down the road. Uh, 11 specific question, uh, didn't mention anything about tolerance. Is that an issue with these long-acting blocks or having to adjust the dosage over time? Uh, as far as we can tell, no. Um, And so, you know, I mean, the way to know that is that with this 40 day thing, for example, to inject it again and see if the duration effects become shorter and shorter. Um, I don't think that would happen because, um, Uh, I'm not aware of any, there's nothing like receptor down regulation or anything like that, that would lead to it. I will say this, in 2012, Sahadev Shankarapa, um, in my group did, um, serial injections with, uh, the sax toxin dexamethasone formulation, which gave a week of block. And he did it 3 times in a row, and each time it was 1 week. So I don't think there's tachyphylaxis or tolerance. So just to follow up on that, then, then when your triggered release, was that depletion of drug that you were seeing over time with that? OK. Yeah. One of the um Pieces of work by Claire that you featured was. Sensory specific block and leaving motor intact. Is there ever any hope of taking it further and Um, I'm blocking just pain fibers, uh, and not sensory, so, so I imagine sensory means like. You can walk motor wise, but you can't feel the floor, um, but it might be nice to have your pain gone, but be able to feel the floor as you walk. I, I, um. I was a little bit of a personal experiment for for Chuck Birdy. I had a, a, a little more than 5 years ago, I had a particularly severe case of of shingles, and, and he was trying sort of everything, although I refused like you to take a narcotic, and And um Chuck said to me, you know, I know it says in the package no more than 2 lidocaine patches, but you can put as many as you want, and I had a very large surface area to treat, so my wife is putting 4.5 lidocaine patches on me, and one day, Driving in, when my tongue got numb, I called Chuck. I said, uh, he said, what are you doing? I said, well, I'm driving on the mass pike. He said, that's not good. Where are you going? I said, I'm going to the clinic. He said, no, you're going to the emergency. I said, no, I'm going to the clinic. He said, all right, take them off now, and um. Um, Tell the nurses, you know, if you drop that it, you know, it's not just a regular resuscitation, but that you need, uh, intralipid to, you know, to, to, to, to treat, uh, lidocaine toxicity. It would be great to be able to treat just the pain nerves. You mentioned neuropathic pain, so after, so I still have that, right? Um, could you ever like, like I, I talked to like neurosurgeons like by doing rhizotomies and stuff, can you select out pain for like ain't happening. Could you ever imagine selecting out Uh, chemically one fiber from another and having long duration effect on just pain but not sensation. So, the problem is that um I actually am not, so in all the sensory selective. Things that I showed you, all I know is they don't feel pain. I mean, they don't feel heat, they don't feel pinch, and I know they can walk. But I can't talk to them. And so asking them, like, well, you could talk to them, they just don't answer. Well, if they answer, it's a problem, um, you know, but or if you perceive the answer, it's a problem, yes. I, I have a clue they actually answer you've, you've, you've actually solved a different problem, get a Nobel Prize, um, but, um. So, there we, we do some proprioceptive testing, but it's kind of hand wavy because it it it We, we don't know whether proprioception is blocked or not. Uh, so, um, and whether teasing out things like fine touch in an animal model is complicated. So, um, with at least with PPX we are now starting to work to do doing a Clinical trial here, will probably happen in, you know, the way it is, it'll be in a year or more. But then, we will be able to answer that using quantitative sensory testing, uh, to really know. Uh, see, I'm handicapped by the fact with that with a lot of things that we've dealt, we sometimes don't, with PPX we think we know how it works. It's certain voltage-gated sodium channels. Um, with the others, we have no idea how they work. Um, so, um, actually, that's not entirely true, but it would take me a very long time to explain it. But to make a long story short, there's many conceptual ways you could have sensory selective block. Um, it could be that you have an effect just on certain sodium channels. Um, it could be that you have effects on other channels, the sodium channels, which are only on sensory nerves, or, and I think with many of these, it's what it is. It's actually hydrophilic hydrophobic balance of each molecule. So you have You know, uh, all axons are not created equal. So, uh, the ones that deal with pain, for example, either have no myelin or very little myelin. The motor ones are large and have a lot of myelin. So, what you want is things that are hydrophobic enough so they can get penetrate down to the sensory nerves and block them, but not hydrophobic enough that they can penetrate the myelin sheath. Around, um, motor nerves. And we think that is how our things work. Um, what they do to, um, some of the other Um, Neurons, we don't know. We actually have a collaborator at UConn who is figuring this all out for us. It's, it's a very complicated story. Well Most important things are complicated. Um, we really appreciate your educating us and your, your, uh, incredible innovations, uh, and long, uh, translational work and, uh, particularly the collaboration, uh, with our department, uh, and, uh, um, across multiple silos institution, um, and I look forward to ongoing collaboration. So thanks very much.