Dr. Daniel Kohane - Enhancing the Performance of Local Anesthetics

Good morning, everybody. Thank you all for coming to this morning's screen rounds. We're welcome today by Dr. Daniel Cohnhan, one of the professors of anesthesia and a senior associate in the pediatric critical care department here at Boston Children's Hospital. He received his MD and his PhD in physiology from BU and then went on to do residency here within the Boston area for both the pediatrics and anesthesia. He then did a scholarship in critical care here at Boston Children's and he currently serves within both the departments of anesthesia and critical care. He has a very exciting lab focusing on research and biomaterials, drug delivery and nanomedicine. And today he will share with us his work on enhancing the performance of local anesthetics. Thank you so much for speaking with us this morning, Dr. Cohnhan. Good morning. I'd like to thank whoever needs to be thanked for me being invited to talk to you here this morning. By way of disclosure, so I'm actually the founder of a company around a drug-looting technology, drug-looting contact lens technology that came out of my lab and also children's holds a fair amount of intellectual property relating to a variety of things that came out of my lab, including many things I'm going to be talking about today. So I don't know which, but it's possible that some of the things I'm going to be talking about today could be commercialized. So as was alluded to, I have the lab for biomaterials, drug-liberated nanoscience, which is another way of saying I have my lab. And so we do drug-liberated biomaterials and nanoscience. And so we started off actually doing prolong duration local anesthetics 25 years ago when I was a fellow here with Chuck Birdie, who's right there. And that has now spread to be a whole bunch of different topics. But today I'm just going to talk about a few of them relating to improving the performance of local anesthetics, which is probably about a third of what my lab does. So it's actually a pleasure to be talking to an audience where I don't have to explain what local anesthetics are and what they're good for and what the side effects are. I usually talk to chemists, which is interesting since I'm not a chemist, but I've learned to fake it pretty well in the last quarter century. And so here they are. We know them. We love them. There's kind of a broad family resemblance from the chemical structure point of view. And even though they are really good at what they do, they do have certain common problems. And in retrospect, one couldn't view the last 25 years of research as being ways of fixing each of those in sequence. So one problem is duration. So most single shot local anesthetics last for less than 12 hours, which in some settings is not enough, like perioperative pain or chronic pain. And so one goal was to produce prolonged duration local anesthetics. And I'll be showing you some of those and also some specialized applications for such technologies. Second problem is that local anesthetics, especially once you can make local anesthetics that last for days, don't respond to the changing needs of the patient. They're either on or off. And so a second major goal was to make local anesthetics, which are on demand, which can be triggered. In other words, another problem which many will be familiar with is that with the sensory block, which is great, comes motor block, which is not always great. And so a major recent goal of my lab has been to develop a sensory selective local anesthetics. So let's look at the first of these, which is prolonged local anesthetics. So a long time ago in figuring out what the design criteria for something like this would be, it was decided that what was needed is something that could be initiated by a single injection. It should be possible for the average clinician to administer it. And you don't have to go to the operating room and get a general anesthetic to start it or a surgeon to implant it. And initially we were thinking that we wanted something that last days for weeks. And that tissue reaction should be good, not too much inflammation. And there should be too much damage to muscle or nerve. Important given the potential toxicity of most local anesthetics, it should not kill you. Generally a good goal. And if it's a drug, if it's a biodegradable drug, if it's a druglery system, it should be biodegradable so that when one of those stops having effect, you could in theory inject another one, that having accumulation of materials at the site. And finally, it should wear off completely. There are other considerations, economic and otherwise, but that's for another time. So when I entered the game, people had already been encapsulating conventional local anesthetics in druglery systems for decades. And basically every druglery system that you can think of has been used, micro particles, nanoparticles, macroscopic devices, polymeric, liposome, mohyogenel based. They had all been used with local anesthetics. And few of them achieved the design criteria that I spoke about, particularly with reference to duration. And one exception, you can see down on the bottom, was the fruit of a collaboration between Chuck and Bob Langard MIT, where they made these polymeric microspheres that contained bupivocane and dexamethasone. And when injected at the sciatic nerve enratt, those would give a duration of block of three to four days and in sheep. They would give a duration of block of eight to ten days. And so the problem with all of these things is that they all suffer from either having the duration be still too short or having the tissue reaction being not good, which was sometimes severe enough that would obviate any potential for clinical use. And to give you an idea of what I mean, here is an example happens when you inject polymeric microspheres with and without local anesthetics at the sciatic nerve. So on the right, you can see what happens when you inject polymeric microspheres at the nerve. These are without any local anesthetic in them. And so there are a pop you can see the ghosts of the microspheres. And you can see the little black dots are the nuclei of the inflammatory cells. So there's some information, but the muscle below looks basically fine, maybe a little bit inflamed, maybe a little bit of a demoness, but the muscle cells are fine. So on this side, you see what happens when you have microspheres that contain eupibircane. And so here you see the ghosts and this maybe a little bit more inflammation, but if you look at the muscle, all of this should look like this. These are shrunken, atrophic and regenerate fibers. And that is local anesthetic myotoxicity. And as we later discovered, encapsulation and sustained release of local anesthetics just makes that worse. And this can become a serious problem. So it was in pursuit of longer duration and the avoidance of this that we turn to site when sodium channel blockers. And these are a class of agents that act at the same, multigated sodium channel. Actually, most of them I should say, as do conventional local anesthetics, but on the opposite end of the of the of the channel. And here they are. They include things like tetra detoxing, which is what kills you when you eat fugu sushi that's not prepared properly. And the sacs detoxins, which is what kills you when you eat seafood with a red tide algae. So this is what we decided to use. And the reason is that these compounds are incredibly potent. So depending on the assay you're using, these compounds are 300 to 10,000 times more potent than conventional local anesthetics. And that really matters when you're using drug delivery systems and you have a finite payload that you can deliver. The other thing is that we had become aware of the fact that the duration of block for most local anesthetics when encapsulated in a variety of drug delivery systems. The duration of block from site when sodium channel blockers is enhanced by co encapsulating, you have to contain dexamethasone and some other compounds. And we're hopeful that we could use drug synergy to get really long durations of effect. So the downside of these compounds is that they're very hydrophilic. So they end up swimming around in your body water looking for sodium channels and they go and they go, oh look, certain channel, but it's the fennic nerve. So it blocks it, but you stop breathing and you die. So there's something similar on the sympathetic nerves, so you get sympathetic blockade and you become hypotensive. But to me as an anesthesiologist, none of my patients breathe. So the first isn't a big problem and hypotension I know out of handle. But importantly, the site when sodium channel blockers don't cause all these things that conventional local anesthetics do cause, and which are either difficult to treat or impossible to treat. So those are the reasons that we went to it to them. So as I mentioned, there's a whole bunch of site when sodium channel blockers and we did all these elegant studies of which one is better than what an therapeutic indices, but we ended up choosing sax to toxin actually just because we could get it in huge quantity from the FDA for free. And so we encapsulated sax to toxin in liposomes. So liposomes come in a variety of shapes and sizes. In this embodiment, they were basically lipid bilayer, rather like red blood cells, but instead of being a single shell, they're like an onion. So it's these concentric shells of water lipid water lipid water lipid. And inside the water phase would be the sax to toxin. And this is worked up by hella fstein barrage. And so we took these liposomes of sax to toxin index mezzone and we injected them at the sciatic nerve in rats. And then we measured the duration of nerve block. And what you can see is that over there is the duration that we got. So from a single injection in a rat, we got a duration of block of a week. The other thing that was gratifying is that the error bars are actually in the graph. They're just so small that you can't see them. And that's actually sort of important when you're telling your patients what durations of block to expect. And facial reaction was good. As you can see from this, this is us opening up the sciatic nerve. You can see it there. And the article residue is sitting right on the nerve where we injected it. And tissue reaction to make a long story short was very benign. There was no muscle injury whatsoever in the group of sax pox and dex mezzone. There was a little bit of inflammation because there's inflammation whenever you inject anything of this sciatic nerve, including saline. Also importantly, the what we did, a tetrazoin blue stained sections of the nerve, the all the experimental groups of local anesthetics were indistinguishable from untreated controls. So we were actually pretty happy that we could get a week of nerve block from this. And that there was no systemic or local toxicity. So there was a downside, however, to this which we will get to eventually. So actually one of them was there was a limit on how much. Sax toxi can pack in without the animal starting to experience toxicity. So a solution that chow zow worked on was to take the site when sodium channel blocker and actually bind it covalently to a polymer. So once it's bound covalently to a polymer, it is inactivated. And the idea was a slow hydrolysis of the bond between the polymer and the tetra toxin would allow it to become free and therefore to cause nerve block. So this is the chemistry and this is what my lab does. So those are mostly actually chemistry lab. And what you can see is you have this long polymer and there's an ester bond, which is what allows the hydrolysis. And then here's that boxy thing at the end, which is the tetra toxin. And that is what would cleave in vivo, allowing nerve block. And so we did sciatic nerve blocks in the rat. And in the interest of time, I'm just going to track your interest to the two panels on the right. And on the top one, the y axis is the duration of block. How long the nerve block lasts with each formulation and on the x axis is the dose of tetra toxin that is administered any truth. And if you look at the red group, which is free tetra toxin in phosphate buffer, saline, you go up to four micrograms at which point the duration of blocks about two to half hours. There is no five microgram group for reasons we'll get to in a moment. If you look at the group with a tetra toxin bound to polymer, the dosing goes all the way up to 80 micrograms and the duration of block goes up to about 72 hours. So three days of nerve block from a single injection. And the reason for the limitation of free ttx is shown down below. Basically, what you can see here is that at five micrograms, all the rats are dead. Because there's too much free ttx on the animals die. Whereas with the ttx that is bound to polymer, you can go all the way to 80 micrograms of dose, which is usually enough to kill 15 rats without any systemic toxicity. And actually, it is so safe that we injected that intravenously into rats and they had no evidence of systemic toxicity at all. So as promised, I'm going to give you a few quick examples of some more specialized applications of drug delivery systems for local anesthetics. So one is application to beer blocks. And so for those of you who are not familiar with it, it's a procedure that is fairly common in orthopedics where the goal is to get a painless limb, which is also provides a bloodless field making operation easy. And so basically what you do is you put an IV, let's say in the hand, and you apply a really tight, tight bandage to the extremity, exagronating it. And then you inflate a really tight cuff, preventing blood from getting back in. And then you take the bandage off and then you inject a substantial volume of local anesthetic into the arm. And so now your arm is pickled in local anesthetic and the surgeon can operate. The patient doesn't feel anything. Actually, they feel the cuff. And so one problem is that the cuff is actually quite painful. And so it is not uncommon for them to convert from volunteer anesthesia or constidation to general anesthetic. The other thing is that in the unfortunate events that the cuff comes down suddenly, you have all this local anesthetic sitting in the arm, which sees their turn of blood flow and says, let's go back where we come from. And basically get this wash of cardiotoxic drug going into the body. So Chris Weldon and Tianjiao Ji in my lab, um, positive that nano encapsulation of conventional local anesthetics would improve performance both from the point of view of efficacy and safety. And the rationale was that nanoparticles being very small and having a very large service area ratio are better at penetrating tissues than our bigger ones. And even if they can't penetrate our better at sticking to tissues. And so the hope was that these nanoparticles would stick within the arm and give beneficial effects. And so these particles were made. You can see these are the micelles, which are 15 nanometers across. That's 15 billionths of a meter. And these are mypizones, which are 100 nanometers across, which is by way about the size that Amazon. I presume. Li-pizones are. And we showed that these both part of types actually slow the release of drug, which ends up being important. And then these were injected into our model beer block. So unfortunately, because of the way rats are constructed, it is hard to do a beer block in their actual extremities. So can jo actually developed a model where he did something analogous in using the tail. So basically point for a male of various. Late die labeled particles or free road meat were infused into the tail. And what we found actually was that the lipozones, which are 100 nanometers, were retained less than free for a shorter period than free die. Whereas the micelles, which were very small, were retained for longer. And those findings actually sort of explain what we found clinically. So when we infused local anesthetic containing solutions, what we found is that the lipozones did not cause any any, any, any tail anesthesia whatsoever after the cuff was taken down. And the micellular formulation lasted about six hours. And that was actually longer than what was obtained with free drug, which you can see was about three hours. And this was true, even though the amount of drug in this curve is five times more than the amount that's in there. And what it is is just that because of the nano formulation, the local anesthetic was retained within the tissues. We also measured blood levels after the cuff was dropped. And what you can see is that with the free food came actually with the light zone, was an immediate washout of the food came into the bloodstream. Whereas with free food came in the so on delay. And with my sealer, food came in the so delayed that actually occur the peak never occurred. So what I suggested is that this approach of nano encapsulating is actually would improve efficacy and also improve safety. Another example of a more specialized use of sustained release technologies and perhaps I think the only demonstration I have here where we have a macroscopic device is a local anesthetic eluting suture, which is also done by Chris. And the rationale is not hard to come by. You know, sutures are a ubiquitous tool in surgery. And once you've been asked if we could make wounds not hurt. And so we made these electrospawn sutures using a polymer that is akin to what is in vicarals. This is a vicarals polyglycolic acid or PGA. And these sutures that we made contains up to 22% bupivocate meaning by weight roughly quarter of the mass was bupivocate. And we showed that even so it was able to retain the mechanical properties that you would want. And this is important because obviously want to maintain good wound, but you want to be so easily you also want to be able to maintain wood integrity in in this figure over here. You can see that wound opposition could be done well and 14 late days later the wounds remain closed on the far side. You can see the histology of the sutures after two weeks within the body. And basically there is what you would expect is a mild macrophaged pedom that in cloud response that looks the same whether it's drug or no drug or vicaral in the suture. And perhaps more importantly there was local anesthesia that was I would say moderate, but long lasting it kicked in at about one day and lasted for about a week. And since then Chris and I have been working on ways to make this work better. So I've shown you a bunch of prolong duration local anesthetic formulations and one possible I mean they're great in the right context, but one possible negative is that they don't change what they do in respond to patients changing needs. So I've been subjected with this formulation that I showed you and let's say it lasts a week and a half in humans and on day three you decide you don't want it anymore. That's too bad. There's nothing one can do. So with that in mind we have turned our attention to making on demand drug livery systems and the inciting event actually about 10 years ago was and it was actually my wedding anniversary. In the morning I went to see my dentist because I had a feeling that since adolescence a huge filling and it was starting to bother me and finally I prevailed on him to fix it. So he took it that he he injected me and I suppose block my superior alveolar nerve and with that he took down the filling and saw this crack running down the bottom of the cavity. So he took out his drill and drilled it down until the crack was gone and then put in even more enormous filling in. And he told me in that wonderful euphemistic way that dentists have that I might have a little sensitivity later that night. So as I mentioned it was my wedding anniversary and my wife and I were out at the Blue Ginger Wells, which I believe no longer exists. And as I was leaving as we were leaving the restaurant suddenly I felt like someone around the spear through the roof of my mouth. And what that was it was the local aesthetics that my dentist injected me with wearing off. So you know I we drove home with me holding my face my wife's driving and I called the dental practice and they made a point for me to see the next day and take motor and I took motor and which didn't really help that much throughout the night. So the next day I saw my dentist prescribed a jar of opioids. So I took the pharmacy prescription filled it and this is actually a key point because in many many procedures this is where patients come and contact opioids for the first time. And so you know I took one I didn't like it. So I continued with motor and it didn't work but you know I could have taken it and liked it a lot and become addicted. You know I could have sold it I could have overdosed on it and by the way a lot of vloggeration local anesthesia work is now being recast as opioids sparing given the broader context of the opioid epidemic. Anyways I modeled through to the next day where I saw an end of don't just who did a root canal which at least stopped the pain and two days later the tooth was removed from from my head. But I remember thinking when I was suffering wouldn't it be nice if when my dentist had injected me and inject me with something that I could turn back on. And that led to this line of research and so the idea is to have something that's initiated by single injection and it's not a systemic treatment is local it's not opioid and the idea it should be at least potentially long lasting but that the patient would determine when they get pain relief. So I'm going to tell you how intense that pain relief is and how long the pain relief lasts and so in the last decade we've come up with a whole bunch of different ways to do this and I'm just going to show you a couple. So in one done by Alina Ray and a similar stuff by Chang Yuzhan we incorporate a tetra detox and and mock you as like it into liposomes. But these are a special life a lot special life zones where the lipids had a lot of double bonds for reasons I'll become apparent in a moment and so the idea is this would be injected by a nerve. And the lipid bilayers also contain a photosensitizer and so when post operatively if you inject if you read the site of injection with light of the right wavelengths which in this case is near red light it makes the photosensitizer produce reactive oxen oxen species which is what photosensitizers do and those would peroxidate the double bonds I mentioned punching tiny little holes in the life zones. And so allowing the drug to escape and to cause nerve block. And so what we showed is that when you inject these things of the sciatic nerve these life zones you get an initial nerve block and where when it wears off if you shine light at the site of injection of where the arrows are the nerve block comes back. So on the y axis you have the intensity of nerve block and on the x axis you have time. As proof of principle this was nice but we wanted to make it better so we did a whole bunch of projects where we try to make the particles more sensitive to light and trying to get better efficacy and also to get more repeatable events. And so Alina Ray did this project and I want to draw your attention to the red line. So in this which is the same technology as the last one only it contained tetra toxin with dex metatomidine this is what you got. So it's worth mentioning that before this we had shown that there is massive improvements of the effective tetra toxin by dex metatomidine. So the idea is every time you shine like a quantum of light and get a quantum of drug release whatever amount of TX comes out is greatly potentiated by dex metatomidine. And so what you can see is the initial injection gave a nerve block that lasted for 40 hours and which by the way is perhaps in some context more than you want. But anyways once it war off if you shine light at the sign of injection which is the sciatic nerve the nerve block comes back and we were able to get 9 repeated nerve blocks of decreasing quality over time. We also showed that we could do something similar with ultrasound and the trick is that we replace the photosensitizer with a sonosensitizer which is does exactly the same thing only the stimulus that works to create reactive oxygen species is ultrasound not like. And this is useful actually potentially because I believe the standard care right now in this country for a peripheral nerve blockade is to use ultrasound guidance. And so what this would allow you to do is use ultrasound to identify where the where the nerve is and see it there in a wrap. Secondly to see where the life of the zones are going because the life zones are actually ecogenic and use the same device to trigger it whenever you want. Speaking of ultrasound Kate this is not triggered drug release but while we're on the topic Kate Cullion who's also in my group has shown that ultrasound itself can actually enhance the effect of local anesthesia. So in this particular project she injected tetrodotoxin at the sciatic nerve at a concentration that was marginally effective. And what this shows is that if she then irradiated the site of injection with ultrasound it also all of a sudden became extremely effective and gave really long nerve blocks. So with all the systems I showed you now there is a they all have basal release meaning they're basically these particles filled with drug and whether you want it or not the drug slowly leaches out. And what that means is that you end up with this initial nerve block which you may not want. And you will also get downstream block potentially that you may not want. The other thing is that if you don't trigger the release still keeps on coming out perhaps at a sub clinical level and that drug is wasted. It would be better to use it in triggered events to get more reproducible triggerable events. So with that in mind way developed this system building on the work by Chow. So here he bound a local anesthetic in this case you usually conventional local anesthetic tetricane he bound it to a polymer. And with a photo cleavable linkage and it's actually symmetric molecules so each one has two local anesthetics. Locals tetricane is very a relatively commonly used conventional local anesthetic and the polymer was Palloxymor 407 which has this interesting property of reverse thermo gelation most materials that we are familiar with like ice and glass actually melt when you heat them. And so the idea is you inject it so it's easy to handle at room temperature as a liquid it's easy to inject but once it actually enters the body it solidifies making a nice deep of formulation. And so the idea is you inject this and whenever you shine light on it the photo cleavable linkage that you see there in blue would be cut freeing drug to. Cold local anesthesia and so these data show that his design work so. PCMT is plus more for seven and then the CUMER NOID linker and then tetricane and what you see is when this is injected actually in the foot pad of the rat not the sciatic nerve if you don't irradiate it there is no nerve lock. Because if the drug is conjugated the polymer is inactivated it can't bind to the sodium channel but if you inject it in the foot pad and then irradiate it you get this nice nerve lock so that is what we're looking for. The other thing that was interesting at least to us was that how much nerve lock you got and how long it lasted was dependent on the irradiance meaning basically the energy in the light that you use and also how long you irradiate and actually if you look at the product of irradiance in time which is the energy density there was a linear relationship between the duration of block and the energy density. And what that means basically that from the patient point of view you can modulate the amount of pain relief you get and how long it lasts by adjusting either the intensity or the duration of the light. So we were pleased with that but the question is could you do this reproducibly so we did the experiment where we injected it in the foot pad and then irradiated that and what you can see is that after injection there was no nerve lock until you shot a light on it. And then you got nerve lock and you could do this repeatedly and a lot of our research on this is now geared towards being able to do this many many times like for days. And moving on to the third topic that I had mentioned. We recently I would say had an interest in making sensory selected local anesthetics and these are a family of compounds that you can change and change that is an animal choir have been working on in my lab. So these are the conventional local anesthetics that I showed you and they're great we love them but they do have certain well recognized failings one is motor block so as I alluded to before the sensory block that you get is accompanied by motor block and this has real clinical implications. So for example for parturians there's a tradeoff between comfort and being able to push and the problem is giving too much as you can actually have arrest the labor. There are orthopedic considerations having to do with the tradeoff between pain relief and having the patient able to cooperate with PT and range of motion exercise after surgery and also early ambulation. And finally there are chronic pain considerations because you can't give people regional or in your axial local anesthetics that would last let's say a week because that would mean paralysis means they can't move and so on. And so to give you an idea of what conventional local anesthetics do in terms of sensory motor function this is what we've been 15 does in a rat and so it gives you a sensory block of 250 minutes a motor block of 220 minutes and the ratio is about one and in our hands, Ropivocaine which is touted as being a sensory selective local anesthetic in humans, whatever. Ropivocaine does about the same thing right. So we produced this compounds, the YK2312, which they raise CMIC mixture. So it's combination two optical isomers. And it gives a block comparable to that of you if you've been able to, but the motor block only lasts 200, sorry, 69 minutes. So the sensory motor ratio is 3.7, which is, to us, fairly exciting. But what is cooler in a way is that, as I mentioned, this is a racemic mixture. If we separated the optical isomers, so these are identical molecules except that one is the right hand. Their mirror image is one is like the right hand and one is the left hand. In all physical chemical properties, like hydrophobicity, charge, everything, they are identical. It's just the head than this is different. And so you can see the first isomer gives a block that's comparable to the racemic mixture. The second isomer however, causes no motor block whatsoever, at least in the rat. In other words, the ratio is infinite, which we were also pretty excited to find out. Okay, the second problem that is encounter with many conventional local anesthetics is local toxicity. And interestingly, in my entire anesthesia residency, this was never mentioned. So I found this out the hard way in animals. And so you can get inflammation, which is not the end of the world, and you can get myotoxicity, which is also, you could argue, not the end of the world though, and condotoxicity, though it can be pretty severe. And then there's neurotoxicity, I mean, actual destruction of nervous tissue. And so we looked at how the compounds we were producing, fair in this domain. And so this is in vitro work where we culture cells and expose them to these compounds. And the cells on the right are C2C12 cells, which are muscle, you know, myoblasts, and that's a proxy for myotoxicity. And these are fiochromosythoma cell line, PC12 cells. And this is a proxy if you believe it for neurotoxicity. And what you can see as a comparison, in both cases, is the black dot line, which is bupivocane. And so we actually produced a whole family molecules, I'm not showing you all of them. Some of them actually fared worse than bupivocane. But in the two that I just discussed, 2312 series, they actually showed no myotoxicity at concentrations that were uniformly lethal to cells with bupivocane. And when we injected them at the sciatic nerve and looked at muscle injury, there was basically what you would see with injecting saline or a local anesthetic. It's really actually pretty hard to see on these low res picks, but biocompatibility was excellent. And then when we did tetrazoleon blue stain sections the nerve exposed to these compounds were indistinguishable from control. By 7.5 millimeter is like 0.25% in bupivocane speak. A final thing, and to me this is interesting, is systemic toxicity. So as I alluded to, the local anesthetics are associated with a lot of systemic toxicity, potentially, particularly the most feared thing is having ventricular fibrelation from bupivocane, for example. And so at the VESTA Bill Clark, who is a member of our department, I sent the two compounds I've been talking about to a company to look at the binding of our compounds to two things, NAV1.5, which is the sodium voltage gaped sodium channel number 1.5, which is the sodium channel that happens in cardiac tissue and which is believed to be implicated in arithmetiasis, meaning the binding of bupivocane to it. And the other things heard, which is a potassium current, which is also in the heart and is also implicated. And when you go for FDA approval of local anesthetics, these are the things you have to look at. And so you can see the two black curves with filled symbols are those response curves for bupivocane. And the those response curves for the two ZYH compound are in open symbols. And so to make a long story short, basically they don't bind these receptors or they bind them much more weekly than does bupivocane, which means that potentially these things are actually really cardio-safe. We didn't design this, you know, this is better, lucky than good. But the other thing that this implies is that these molecules are some of them receptor specific. So this is a receptor specific effect, not just a non-specific effect. And finally, one problem with local anesthetics is that they are a relatively short duration. So in another member of the ZYK family, when we injected the sciatic nerve, we got a block duration of 550 minutes at the same concentration as the bupivocane. So it's roughly, it's actually more than twice the duration of effect as bupivocane. And actually to boot, it's relatively sensory selective, they're not absolutely so. Well, I thought, so this is actually one of two, an antimerism was really interesting to me from a molecular point of view, is that if you look at what the other an antimer does, it does absolutely nothing. Okay, so these are two molecules that are mirror images. One has this ultra long effect and the other one does absolutely nothing. And this is gonna keep us busy into the foreseeable future, trying to figure out why this is so. So in summary, what I have shown you today is that basically local anesthetics can be made to do a lot of things that they are not traditionally thought of doing. They can be made to have very long durations, either free or encapsulated. They can be made to be patient responsive or undemanded. They can be made to be safer. And that can be done either by synthetic chemistry or by encapsulation. And they can also be made to be sensory selective. A huge number of people have contributed to this work, not all of whom are in this picture. Apparently, this person is a resident who is in here, but was born to my lab and has now an anesthesia resident. And you can recognize a lot of our fellows. There's Michael Woodruff and Brian McEllivan and Kate Cullian who are all now faculty here or elsewhere. And he's not really my lab, I was in kids. And this work has been supported by the NIH since 1999. And I've been, this work has been supported by the NIH since 1999. And also we've had the support of my division. And this may sound really trivial, but it's not. One of the main forms of support they gave me is leaving me alone. So I would go to my lab and pull up my drawbridge and do my research and Jeff did not put me on committees and all that kind of stuff. And so I just did my stuff. And also the support of my department. And when I say support, I remember having a conversation with my chairman, I was at the MGH, and he was following me down, I support you. And I actually can't believe I said this to him, but I was young. I said, sir, I actually used his name, but I'm the computer. I said, sir, support has a color. And that color is green. And without the departmental support that I've received, I'm not sure I could have achieved what I have. And my group. So thank you for your attention. And if there's any questions, I'll be happy to take them. Thank you. Thank you. Thank you. Well, thanks, Stan. I'd say, wow. I mean, we have lots of these combined surgery anesthesia grand rounds. And sometimes it's more interesting to surgeon. Sometimes it's more interesting to say, I'll just, I think this is one that really is of equal interest to all of us. I've always looked at local anesthetics just kind of like, whether you're the doctor or the patient, kind of like magic. Like you inject this little needle in somebody, and then you cut them and you can be talking to them. They don't feel it. Like that's just crazy. Still every experience I have. But the history and this department of moving this forward is spectacular and all the people who have contributed to dance work. So questions, comments, there's lots of experts here in the field. Dan, that was spectacular. And as many people know, Dan, and I have collaborated on other work that you touched on barely here. And the challenge with all is getting it into clinical practice. Can you comment a little on the barriers to getting through commercialization and approval in this space? And I think for anesthetic drugs in general, compared to tablets that you take twice a day for chronic diseases? Well, actually, you are far more expert in that than I. So I would defer to you. What I would say is that the barriers are huge and they are as subtle. So for example, I think most of us are relatively pure researchers. And the thing is that if you want stuff to get out there, you, for example, have to some degree orient your research to what is patentable. Because ultimately, if things are going to get out there, this may sound crashed. It's nonetheless true. Someone probably has to be able to make money out of it. And without a patent, that is difficult. And there's a whole sequence of things that are needed for commercialization. There's a whole bunch of regulatory things, which are really important. But we are not at all trained to think about certain kinds of toxicity studies, certain kinds of animal studies. All these things have to be done that are drugs to build. You're over two years or some finite period. All these things that go into just getting the FDA to allow you to test in humans. And then there's the whole aspect of doing serial clinical trials, getting investors to put money in. And answering a question would actually require a seminar series. It is a, as you know, it is a big deal. And I think one point is that for a company to invest that amount of money in it, they have to make enough of a profit to be worth risking $100, $200 million. The economics of it are the average person in the US has roughly perhaps six surgical procedures per lifetime, depending on estimates. That would be at most six doses, and your ear tubes are not going to get a dose. And other things aren't going to get a dose. And so even if millions and millions of people have operations a year, it's not hundreds and millions of doses the way Prylasek is hundreds and millions of doses. So that for a company, the incremental benefit per dose and per procedure has to be enough that they can charge more than bupivocane for it. And that has been a barrier to commercial interest. We've gotten, Dan and I together, are having been involved with companies that have taken things into clinical trials. But the barrier to getting big investment and the biggest companies in it has been that kind of financial thing. When Paul, Mark and I and others were anesthesia residents, there were new drugs coming out all the time in anesthesiology. There have been remarkably few in the last 15, 20 years, just because of that financial barrier to it being enough better in pharmacoeconomic terms from what's there now. I'll just say, though, the one thing I disagree is that, there actually is a place for local anesthetics with the ear. So we actually have developed formulations to numb up the ear drum effectively for procedures or actually to numb up the, we have a system I don't know the time to get into that will actually get the drug across the tympanic membrane into the middle ear to treat otitis media. And if that works, it would be potentially a huge market. I want to go back to the video of getting ear to which are in the scope of mass care. No, but I'm saying three years with otitis media, this way we'll get a local anesthetic. A fabulous work done in terms of advancing new materials and new formulations. I think related to Chuck's comment in terms of translation is this whole challenge of just tremendous biological and biological variability in potential patients for new formulations. So when you're designing studies and you're planning experiments and you have to take that into consideration, when you're looking at combined drug action interactions of different local anesthetics, how do you actually plan those studies to be looking at performance, enhancing performance, when you're looking at something like sensory nerve black to determine whether or not there's synergy, whether there's additivity, and how those study designs and experiments somehow play out with potential translation. So there's two different answers, both of which are actually of a statistical nature appropriately enough. One is that I've decided arbitrarily about 20 years ago that I was not interested in anything where the effect size is not at least a 20% change. So I always power accordingly. And so in terms of just a p-value, I'm all set. I know what number I need, usually. And then the problem becomes when I start using combinations of drugs, how do you actually know how they interact? And so the issue, the buzzword is synergy, which, by the way, partly in my nanoscience universe, is a word that is incredibly overused. People do, like, drug A has effect X. Drug B has effect Y, and A plus B has four times X, and they say that synergy. And it actually isn't, even though it looks like it. So to really show synergy, and we're not going to get into this, you have to do those response scripts for each one and do this nightmare thing called isobulogram, or a combination index. We're talking about a huge amount of experimentation to really formally show, and you've done this with me, synergy. It is not for the lazy, I was going to say fiend to part, but it's a lot of work. Thanks so much, Dan. This is so awesome. I just wanted to ask a quick question. I think when you talk about the philosophy of local anesthetics, you would say the ideal local anesthetic would be long lasting, non-toxic, sensory selective. But if you really were to think about the ideal local anesthetic, it wouldn't be completely sensory dense, because we would all say it's actually unsafe to have somebody be completely sensory blocked. We want them to have no pain, but we would like them to be able to sense enough so that they are safe when they leave the hospital. I'm wondering, when you designed some of your trials, et cetera, is there a way using your models to understand the degree of block? Because we would like a long lasting block, but we would like it to be a block that blocks pain, but not complete sensation. Does that come into some of what you're doing at this point? Yeah, so the concern is that you have your arm numb, and let's say you can move it. So you're leaning like this and smell something burning, and you realize that actually you are leaning against the stove. You left your hand on, and it's your hand cooking, and things like that. And so one thing which we're going to address, and I don't know, is for example, with these sensory local anesthetics, do they actually block proprioception? I don't know the answer to that question, too. It's something we're looking into. The other thing is that it is possible with many things that by hitting the infusion rate just right, or the dosage just right, you could have it where it's not a complete block. And we know, I didn't show the data, for example, with the triggerable systems, you can, for example, hit the nerve with a inside the body, with near and for headlight, with an irradiance that gives you half block. You know, it's like the constant like two twitches with local anesthetics, with a muscle accents. You like, you don't have full block, you don't have no block. You're somewhere in the middle, and the patient would have a light emitting diode where they can dial in exactly what they want. That is the best we can do right now. Well, time is short. I want to point out that one of the most important things that I see here is the lineage of mentorship. Dan still listing Bob Linger on his slides. A quarter century later after being in his lab, and then every slide he put up, he said who did the work, which we all know great scientists do. But there are quite a number of people in this room who have gone through this experience who benefit from it. And this invitation for an anesthesia-quil care chemist, first, actually came from the Department of Surgery. And in part, that is because of mentorship that he has demonstrated to us. A bit of a lot of people in this room didn't know that Chris Weldon did basically search in Dan's lab because Chris does some of the things that are prominent in many other ways. But Chris had been working on this thing for a long time in a Dan's mentorship and with incredible support. And I'm a Guire who we mentioned is actually a surgical resident who came here to work with Polidicin myself, but wanted a chance to learn real basic science. We've posed to several different options, and I think she didn't make a mistake by working with Dan. So I want to thank you for the mentorship you provided to so many people. And the great work this is going to magnify them and have this dream come true. So thank you very much. Thank you, sir. Well, thanks for inviting me. There was nobody sleeping. There was one guy. I'll find him. I'll call that, mate. He was probably on. Yeah, yeah, when when Judas spoke in here, I'm sure you sign speak sometimes. There's two things are amazing. One is like, it was standing around. The two of you look around and it was never anybody's. Yeah, and that's the that's the mark of a great speaker in it work. So thank you so much. What was your impression of the Israeli images? Matters? What was your impression? No. And it also was for you. Thank you. interested in this. Answers from outside. Yeah. Okay. I'll tell you about the entry in the room. I'll tell you about the entry in the room.