 So, good morning everybody. Thanks for coming. I feel very honored to introduce Dr. Enrique Rodrigues-Poulin today. We are very lucky to have him here today. He is very busy and traveling a lot. He is an outstanding scientist who is famous for his work in cell biology and in RPE and in epithelial biology and let me briefly tell you a little bit about his academic career. So, he received these from Argentina and he received his MD, can you hear me all? Or shall I talk through the microphone? It's okay. Okay. So, Dr. Rodrigues-Poulin is from Argentina. He received his MD from the University of Buenos Aires and then after three years of training in biophysics he transferred to the United States to NYU in New York and trained there as a cell biologist. And during that time as a postdoc and subsequently as when he moved into assistant professor position he co-developed the MDCK model and used it to study how epithelial polarity is established and maintained. And for example one of his really key papers is that his laboratory discovered in 1978 that enveloped viruses can be butted from MDCK cells in a polarized manner. So, this was one of the first papers that showed that molecules can be expressed and located, localized in a polarized manner. He then became associate professor at Weill Cornell Medical University Medical College and this was in the department of cell biology. He also became Joseph Hinze professor for cell biology and then subsequently he was appointed as a Jules and Doris Stein professor in the cell biology department of cell biology and of thermology, excuse me. And then he was appointed as a scientific director in the Margaret Dyson Vision Research Institute. And since then he has been appointed as a Margaret Dyson professor of atomic research in this vision research institute. He's also professor of cell and developmental biology and professor of neurosciences now. And he also was director of the vision training grant in the department. He has received many awards and honors and for example the Irma Herschel Award, the Bensley Outstanding Young Cell Biologist Award and the Established Investing Award of the American Heart Association. And we originally had this visit scheduled in November but he couldn't come unfortunately for us but fortunately for him because he received the highest award that you can receive as a scientist in Argentina, the RISE Prize. I hope I pronounced that correctly. He has been funded by NIH for over 30 years. He has published 194 publications so far and I just want to emphasize that his research is really, he has discovered many key processes in apical basal sorting and trafficking and epithelial cells and he has made many very exciting and interesting discoveries in RPE cell biology. So this is very important especially for macular degeneration. And I stop now and let you talk. Okay Sabina, thank you very much for the very nice introduction. You have a pointer. Yes. Okay, so this project started a few years ago in the lab and it's part of our, my intention to do something that is a bit more translational. Most of my work has been at a very basic level, basic cell biology level, the cell biology of epithelial cells, epithelial polarity. And in the lecture I'm going to give later today, I'm going to deal a little bit with that. But this one is more focused for this audience on the problem that we have been interested for a while, which is the problem of lipofusing. How lipofusing damages the RPE, how lipofusing causes blindness and if this is possible or not to get some kind of therapy for these type of diseases. So this by the way is where I work. This is Wild Cornel Medical College. This is Rockefeller University. This is the East River. Sloan Kettering Central Park. And this is where I live. Okay, so. So this is a monogenic disease usually caused by mutations in the APCR4 transporter. And there are large amounts of lipofusing deposited in the RPE and causes macular dystrophy and blindness by about the fourth decade of life. And this is a clear example of this disease that lipofusing is bad for the eye. So what is lipofusing? We're going to go into that. In terms of macular degeneration, lipofusing accumulates age in all of us. With age, we have an increasing amount of lipofusing. And by the age 70 and above, we may have up to 20% of the cytoplasm occupied by lipofusing. But whether this is a factor or not in the etiology of AMD is not clear. The work by George Hyman and others have clearly shown that AMD is linked to a variety of genes that can predispose to AMD. But lipofusing in particular in APCR4 is not particularly linked in that sense. However, there's the possibility that lipofusing could somehow contribute, be one of the contributing factors to develop the disease later on in life. So what is lipofusing? Lipofusing is a fluorescent material that accumulates with age in the RPE. You see this in the section of the retina. This is the RPE. And when you look at the cell loaded with lipofusing, an RPE cell, you see all of these dotted elements in the cytoplasm. And those dots are lysosomes. So the lipofusing some material accumulates in lysosomes in the RPE. And it also accumulates in many organs, most organs in the body with age. But the lipofusing that accumulates in the brain, for example, or in the liver is completely different from the lipofusing that accumulates in the eye. The celloid lipofusing that accumulates in the brain, for example, is mostly based on protein. The lipofusing in the eye is mostly based on lipid. So we know very well that from the work of young that photoreceptors are continuously regenerating. And it takes about 10 days to move material synthesizing the inner segment to the tip of the outer segment, where it's then phagocytosed by the RPE cell. And in our lab, Sylvia Fineman discovered one of the two major receptors in RPE phagocytosis, the alpha-V-beta-5 integrin. And with Marti K, these two receptors mediate this daily phagocytosis of outer segments into phagosomes, which are then converted into phagolisosomes and degraded. The material, the outer segments degraded there. Now the lipofusing is a consequence of this activity, the activity of phagocytosis, and the activity also of the visual cycle, which is a cooperation between the photoreceptor and the RPE that leads to the production of this retinoids. This is shown in this slide in a little more detail. So we get from the blood 11c retinal, which is converted into 11c retinal and shipped to the photoreceptor, and it's a prosthetic group of rhodopsin. So when light hits rhodopsin, 11c retinal is released and converted into old trans retinal on the inner side of the sac, in the photoreceptor of the membrane sac. And then ABCR is a free-pace that transports old trans retinal to the cytosol. Here we have, the old trans retinal is relatively toxic, so there's a mechanism to reduce it throughout dehydrogenation to old trans retinal. The alcohol is less toxic and it's moved to the RPE where it is, through a series of steps, isomerized back to 11c retinal and shipped back to the photoreceptor, and so that's the visual cycle. Now, a branch of the pathway is the following. Part of the old trans retinal doesn't follow this route, but follows an alternative pathway and becomes dimerized and linked to phosphatylethanol, informing these retinos, which have these shapes. And the process is completed in the lysosomes of RPE cells, and these components are relatively very stable, so they can stay for years. They are quite resistant to all known hydrolysis, so they are very difficult to deal with. It's sort of like a lipid diamond, if you wish. Now, under, in Stargardt disease, ABCA4 is mutated, and so this pathway, this alternative pathway is enhanced enormously, and therefore we have an enormous production of these viscretinoids. And the accumulation of these viscretinoids is actually quite toxic. For example, if you can incorporate them in vitro, you can synthesize them, and in this experiment what we did was to synthesize an A2E or the old trans retinal dimer to different viscretinoids and incorporate them into the cells by exposing them to this viscretinoid. So, at concentrations that we know result in intracellular levels similar to those of an aging eye. In those concentrations, we can see that there's a clear effect on viability of both these RPE cell lines and the human fetal RPE cells. So, the objectives of this project in our lab are to develop a drug that would remove lipofusing from RPE. Then, if we obtain it, we would like to see whether this drug can stop stargon disease in humans. And of course, if we would come to that point, it would be interesting to see whether it has any effect on patients with AMD. So, this is A2E, and the way that we started looking for a drug that would bind into it was to use a property of these viscretinoids. These viscretinoids are fluorescent, and the fluorescence, this is the fluorescence in water of A2E, for example. The fluorescence of these viscretinoids enhances when you place them in a medium, in a nonpolar medium. For example, this is the fluorescence in methanol, this is the fluorescence in DMSO and in exane. So, the most, when the polarity is decreased, the fluorescence is increased. So, then we screened drugs that would cause an increase in the fluorescence of the viscretinoids. And one drug that came to mind right away were cyclodextrins, for several reasons. Cyclodextrins are native compounds. They are formed by several units of glucose linked so that the core, the inner core is hydrophobic, whereas the external surface is hydrophilic. So, we have the alpha cyclodextrin has six molecules of glucose, the beta has seven, the gamma has eight. And this is the core that I showed you before, the fluorescence of A2E in water up to exane here. And this is what happens when you expose, when you put A2E in the presence of different cyclodextrins. What you see here is that in the presence of alpha cyclodextrin, this practically no increase is the green curve here. With gamma cyclodextrin, the increases are bigger. And with beta cyclodextrin, you have the highest increase in fluorescence. So, this suggests that the hydrophobic arms of A2E, which are fluorescent arms of A2E, are being encapsulated within the hydrophobic core of the cyclodextrin. And you can use, you can see what is the fluorescence that you get in the reaction as a function of the concentration. This is micromolars of the different cyclodextrin. So, you see that clearly the one that has the highest increase in fluorescence is the beta cyclodextrin, which is relatively not very soluble than the methyl beta, which is more soluble, so you can use it at higher concentrations. The gamma, you get fluorescence at the higher concentrations of cyclodextrin and the alpha, of course, you don't get any fluorescence. So, in a paper that we published last year, we studied different properties of the interaction of bifuridinols with cyclodextrin. So, this is a demonstration that when you incubate, you know, when you expose bisretinoids to light or to an oxidative environment, the typical absorbance spectrum of this bisretinoids. This is not fluorescence, but this is absorbance. It has two shoulders corresponding to the two arms of A2E. If you expose it into photoxidation, you lose that spectrum. You lose the two shoulders. If you incubate the A2E in the presence of alpha cyclodextrin, you don't protect against the loss, against the effect of oxidation, but beta protects very well. Gamma has an intermediate protection. And the same is true for auto-oxidation. Oxidation just by leaving the A2E in a tube for a week. For example, you lose the shoulders and alpha doesn't protect, beta protects very well and gamma, intermediate protection. What we did was then to model the interaction between cyclodextrin and A2E. And this was work done in collaboration with Harrell Weinstein, who is an informatics. He's the chairman of the physiology department and an expert in this modeling technique. So, he helped us with it. And this is how A2E looks. It has these two arms and the short arm, the long arm. And it has a size, a diameter at this level of about, the maximum diameter of about 6.5 Armstrong's. And here you have the inner diameters of alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin. So, alpha has an inner diameter of 5.6, so it's clearly too small to allow penetration of the arm of A2E. Whereas beta cyclodextrin has an inner diameter of 6.8 Armstrong's, smaller one. These are toroidal molecules, so they have two, on one side the diameter is 5.6, on the other side is 8.8. For beta it's 6.8 and 10.8. So, this clearly has the right size to accommodate A2E. Whereas gamma cyclodextrin is a little large, so the arm enters into the ring, but sort of it's not very tight. So, and this modeling work told us that we can get even one or two molecules of cyclodextrin, one per arm. And all of these configurations are possible from the modeling point of view. And one prediction is that beta dimers would bind A2E with higher affinity. And actually this work done by Ronald Breslow, who is a very prestigious chemist at Columbia University, he's a member of the National Academy of Sciences, who several years ago published a very interesting paper in which he studied the interaction of cyclodextrin with different types of substrates, either monomeric substrates or dimetic substrates, some of them V-shaped like the cyclodextrin. And this work clearly showed that, and this was experimental work of very, very high quality. They produced and tested a whole variety of different cyclodextrin. And demonstrated that some cyclodextrin, sorry for it, I keep having problems, some cyclodextrin, when you make cyclodextrin like this, a dimer with a fixed angle and so on, you can get affinities which are up to, you know, tend to the affinity of an antibody for an antigen. So we have eventually the potential of developing very powerful cyclodextrin in the future, and we have been talking to him about going this route. Now, we don't know if affinity is an important, it's important to have such a high affinity. That would be helpful for the drug effect or not, at this point. I want to emphasize this point. So what an experiment that Marcelo Nociari, who led this project, did with Guillermo Lehmann, was, as I told you, we can load RPE cells with A2E. And then when you do microscopy on these cells, you can see this type of pattern, which is typical of lysosomes. So you have these yellowish fluorescence of A2E. And when you incubate these cells, in the presence of a cyclodextrin, you can remove a good amount of it in over a period of 24 to 48 hours, about 50% removal on average. And you can measure that by quantitative microscopy, or you can measure also this by HPLC, which we did in collaboration with Roxana Radu at UCLA. Also, the other experiment that we went, one step ahead, was to do the removal experiment in excised eyes from a mouse, which is a mouse model of a Stargard disease. So it's a mouse that we got from Chris Polchevsky. This actually is more than Stargard, because it has a double mutation. It has a mutation in the ABCA4, and it has also a mutation in the dehydrogenase. So it's a double knockout mouse. And these mice accumulate very large amounts of lipofusing in the RPE. And so what we did was to take the eyes of these mice, enucleate them, so cut them in half, then take the back half of the eye, peel the neural retina, and expose the eye cap, the remaining eye cap to the cyclodextrin, and then we shipped within measurement, quantitative microscopy and HPLC. And we saw that in both cases, about 40-50% removal over a period of 72-hours treatment measured by a little higher with HPLC. And the removal was... These are the different peaks that you find, different these retinoids, A2E and old transredinal dimers are the most abundant ones, but they are smaller ones. And it was pretty effective for all of them, the same efficiency for all of them, the removal rate. And we were trying to show an effect in vivo. Here the issue was what is the best delivery that we can use, the delivery route we can use for the drug. So after talking to several people, we decided to go to use the intravitrient injection of the cyclodextrin, simply because it had been tried for other drugs, and so testing other routes was not so easy to think at this point. Anyway, so this is not so easy to explain. That was a work of Guillermo Lehmann in the lab who is very good with his type of procedures. And he injected in one of the eyes in the mouse, the cyclodextrin, and in the other eye, buffer. And we shipped the eyes to Roxana Radu, who can measure the cyclodextrin, just one mouse eye. So the HPLC sensitivity is good enough to measure all of this in one eye. And so this is comparison of pairs of eyes. In every case, we saw about 25% or 30% removal after these type of protocols, several injections in a week. And it was efficient for several different B-thread denoids. Now, based on these initial observations, we were invited to submit a proposal to this newly created institute, Transitutial Therapeutic Discovery Institute, in which Michael Foley is the director. And this is a new initiative in Cornell, in which people who have, researchers who have a project in the so-called Death Valley status, meaning you have a drug, but you have an interesting effect, but you are very far from having a drug that you can use in humans. You need to go through many different steps in between, and Greg can tell you about this very well. So the question is, this is a situation in which, we consulted several different companies and they said, well, it's a nice band. You need to have more. You are not yet, in terms of convincing us that this would be useful in clinical work. So this was a good opportunity because this institute selected our proposal together with a proposal by Fred Maxfield in our institution, who has been using cyclodexins to treat, or to develop a treatment for nemenpic disease. Nemenpic disease is a disease in which kids accumulate cholesterol in lysosomes. They cannot get rid of cholesterol because they lack a particular cholesterol transport. Nemenpic C protein. So, and that causes death very early in life. And there have been some pretty good successes in working, for example, with cats that develop this disease. Doing intrathecal administration of beta-cyclodexins prevent them from getting the disease. So now they are using this, people are using cyclodexins to treat some kids who develop the disease and there's a foundation, a precision foundation that supports this because I mean the kids are actually precision. They are in the family and they're desperate to get some kind of treatment. So this has been, and some of the cyclodexins are actually FDA approved. Cyclodexins are used as delivery vehicles for drugs. So in a way, that's already a step that has been crossed. And then finally, the collaboration in this case involves putting a group of Japanese chemists from Takita Pharmaceutical in a space in Cornell and then helping us develop different derivatives of cyclodexins. So what is, how can we make cyclodexins more powerful? Well, A2E or lipofusine accumulates in the lysosomes, but so the cyclodexins has somehow to get from the outside to the lysosome. And the conditions that we are using them now, they are getting to the lysosome through pinocytosis or cell drinking. That means it's a very inefficient process. Now the cells, all cells in the body have this type of conveyor belt to transport lysosomal hydrolysis secreted into the medium that have minus 6-phosphate, they bind to minus 6-phosphate receptor and by a process of endocytosis, they reach the lysosome. In principle, one can get a speed of about 100-fold or 1,000-fold bigger using this than the regular pinocytosis. This is Fred Maxfield, who is interested in getting better cyclodexins for the treatment of pneumatic disease. So because we were going to get several different tubes every month to test, we developed a more high throughput type of assay to measure removal of lipofusine. And this is shown here, so we use CHO cells, which are very easily loaded with lipofusine. So we load them, the lysosomes are loaded with lipofusine. Then we expose them to cyclodextrin for 16 to 20 hours and we stain them with lysotracker for the lysosome. And then we can do quantitative microscopy of the fixation, making the ratio between lysotracker, which marks lysosomes and A2E fluorescence. And so I'm going to show you some of the drugs that we tested using this assay. So this is alpha-cyclodexin. As you know, this is too small and doesn't have any efficient removal. It doesn't remove A2E from the cell. This is the one, the cyclodexin that we have been using so far, beta-methyl-cyclodextrin. It has a removal effect, a concentration of about 500 micromolar. This is the Amono-Amino cyclodexin that is used by the chemists for the first step in the production of the other cyclodextrin. This one doesn't have a very efficient removal capacity. This is one cyclodexin in which a Manosyx phosphate was added as a ligand to the drug. And here you have a removal capacity at least 10 to 50 times higher than the original cyclodexin. So now it's in the order of the micromolar range. This is another variation of Manosyx phosphate cyclodextrin, also pretty efficient removal. A different variation of Manosyx phosphate, also pretty efficient removal. When two Manosyx phosphate tags were added, the removal was sort of not very good, so this is not helping the process. And so by using now the fluorescence of the A2E phosphate, we were able to measure the affinity of the cyclodexin for the particular A2E. And so this is what I showed you before. The fluorescence of A2E in the presence of alpha, beta, gamma, or the difference Hp, beta, or m beta cyclodextrin, different cyclodextrin. And these are the affinity. Methyl-beta cyclodextrin, for example, is the one that we have been using. It's in the order of 10 to 7 times 10 to the minus 4. So it's in the order of micromolar and high micromolar range, low micromolar range. Addition of Manosyx phosphate actually decreases the affinity. The Kd goes higher. So even when the affinity is decreased, we have a much higher removal capacity. And this is probably because we have the ability to get the drug faster into the lysosomes. And so one interesting observation here is that this acido, some product that came up from this screen is an acido cyclodextrin. This one has a pretty high affinity compared to the other ones. But it doesn't have Manosyx phosphate that however can remove very efficiently, almost as efficient as the Manosyx phosphate. So we're interested in this product as well. So as I mentioned before, it may be possible to get cyclodextrin that have an affinity of 10 to the minus 11, a Kd of 10 to the minus 11. Again, we don't know if that would be good or not. That's something that we have to test. And that's because we don't know what is the mechanism of cyclodextrin is using to remove the lipofusing from the cell. So in conclusions so far, a single Manosyx phosphate tax improved considerably the potency of CDs to remove A2E. Introduction of Manosyx phosphate tax reduces 10-fold the affinity of CDs for A2E, and nonetheless they are still much more efficient. And in some cases we see that higher affinity of CD for A2E correlates positively with removal potency, in the absence of Manosyx phosphate tax. So clearly what we would like to do in the future is to understand more about the mechanism for function of cyclodextrin. How do they penetrate the lysosomal compartment of A2E? Do they promote lysosomal exocytosis? Lysosomes are not immobile organelles. They are very dynamic organelles. And now in the last few years, the work by Andrea Balavio in Naples has shown that lysosomes have a tremendous flexibility in terms of response to different situations that the cell has to respond to. So we see that the cyclodextrin, we don't really know how they work. I mean one possibility is that the lysosomes may be now fusing in higher amounts with the cell surface and removing this to the exterior. Or another possibility is that somehow the cyclodextrin are promoting transfer from the lysosomal compartment to a different compartment in the cell where the lipofusing can be degraded. And actually in that sense, there's an interesting experiment that was reported. We were at some point interested in studying how I mean thinking, could we get some kind of hydrolysis that can be degraded to be lipofusing. I told you that it's very difficult. And one idea that we had was maybe we can go to an expert, a microbiologist who has a lot, a collection of different bacteria. And since there are some bacteria that can be degraded, oil, it may be possible to find some bacteria that would degrade lipofusing. And actually we had already made a contact and a paper came out and someone else had the same idea. And actually Janet Sparrow collaborated with that work. And interestingly they found that some peroxides can degrade lipofusing. And this is interesting because peroxides are concentrated in the peroxisomal compartment in the cell. So one possibility is that somehow in terms of mechanism of action is that the cyclodexins are promoting transfer out of the lysosome to peroxisomes where they may be degraded faster. So that's a clear possibility for the future. Anyway, so what we need to do next is to find a way to study removal, not only removal of lipofusing from the eye in animals, but also to restoration of vision. So that would be the next step. Can we restore vision? Can we prevent loss of vision by this treatment? And there are many issues, administration route, then with developing of optimized cyclodextrin, et cetera. And we have to consider in this is that the Manus X-phosphate receptor in most cells is expressed on the basolateral surface of epithelial cells. So in the case of RPE, the separating cyclodextrin from the vitreous would have to get first to the apical surface of RPE through the neural retina. And we don't have a receptor there. So that would be a good route of administration in that sense. It would be better to come from the scleral side in that sense. And this localization of Manus X-phosphate in RPE is clearly basolateral in the lysosomal component. And this is a co-distribution of Manus X-phosphate with a basolateral marker in RPE. So, again, another view of the basolateral localization in lysosome. So in terms of in vivo experiments, we started a collaboration with Glenn Bruski who's a vision physiologist working in a difference in the center for visual restoration in Burke Institute, which part of Cornell University in Westchester, a little bit upstate. And so he developed this technique to measure vision in mice, auto-kinetic tracking, as you may know, we have heard about. So in this technique, the mouse is placed in a platform in the center of a virtual cylinder that moves around the mouse with stripes and the stripes can vary in intensity, contrast, speed, and so on. So you can measure two parameters called spatial frequency and contrast sensitivity. So these are the DKO mice at 200 days old, 600 days old. We can see that there's a continuous increase in lipofusing in the eye. And at 700 days old, that's when we see that the first evidence of damage to the RPE cell. This is almost two years old mouse. Up to this point, there was no damage noticeable to RPE cells, but at this time we see that the RPE cells become larger indicating that many of the cells have died and the other cells are occupying the space. Interestingly, when we do the visual function tests, what we see is that you know, using spatial frequency as a parameter, the vision of these mice is normal up to about 600 days. And the case, very drastically, between 600 and 700 days, these would be the control mice. They have a much more gradual degrees of vision at this point. In terms of contrast sensitivity, these mice, these are bad parts of this. We had accumulated all of these mice over three years and so we were able to send them mice of different ages so they could do these measurements, but obviously this is not an easy model to work with. If you have to wait 200 days to 700 days to do the experiment. So there are ways to improve the model. We think that if we would expose the mice to more light and probably they would develop regular levels of light, because the levels of light that you have in the mouse room are pretty low. You would have a faster development of this. Anyway, so also we saw that there are some loss of contrast sensitivity with age. These are mice of about 300 days old and 380 goes here. So this shoulder disappears after 300 days. So this is a little better. It's one year instead of two years, but still pretty long in terms of looking for a model. Our first experiments in trying to inject intravitrally the drug into mice didn't show it was just one injection and looking seven days later just to do some trials. We didn't see so much effect in the special frequency, but we saw a small prevention for three mice in the decrease of the contrast sensitivity from here. Instead of one here, one here for three mice. It's a very small effect and it's also one initial trial. So in conclusion, using a screening acid for small molecules that interact with A2E, we identified beta-cycloidextrin, which has small oligosaccharide rings with a toroidal hydrophobic cavity. Beta-cycloidextrin, but not alpha or gamma, prevent quenching of A2E fluorescence by water and prevent their photo-oxidation. The protective function of these beta-cycloidextrin originates in the ability to encapsulate the hydrophobic arms of A2E into their hydrophobic cavity. Beta-cycloidextrin can remove A2E from our p-cells, but the mechanisms are still unclear. Syncl-6-4 post-weight tags improve considerably the potency of CDs to remove A2E in vitro. In the future, we would like to test beta-cycloidextrin and improve derivatives for recovery or prevention of vision function in the KOMIs. Eventually, if this works, we would like to test them in start-up patients. A form of beta-cycloidextrin is already FDA approved and is being used to treat nemenpic disease. If beta-cycloidextrin is effective in start-up, obviously it would be interesting to test them in patients with atrophic AMD. Marcelo Nociari and Guillermo Lehmann did a lot of the work reported here. Chris Barnes, a graduate student, also participated in the last group of experiments with nanosexphosphate. Collaborators, I mentioned, Fred Maxfield had Weinstein. Cilar Kiss, who advises a clinical assistant professor who advises with star-grade disease. David Warren and Matthews helped with the PNAS paper as the chemistry-cycloidextrin. We have a patent filed and published in August 2014. We received funding from all of these places. And this is the Trinstitutional Discovery Institute with Michael Foley and the group of Takeda chemists who have been helping us. Thank you very much and I'm glad to answer any questions. Can we have light? Yeah, you mean kinetic model to study binding? Yeah, for the binding, we study the property of fluorescence of the cycloidextrin and the fluorescence increases when they are sort of better protected from the water by the hydrophobic cycloidextrin. We didn't go beyond studying the KDE but what we are interested in doing is really trying to see whether to understand more about the biology of the system because we don't really have anything about the removal process. Clearly, they are not there anymore so they disappear. So they cannot be detected by HPLC in the eye after you apply the cycloidextrin. So where are they going? Where did they go? They go out, so we are trying to figure out if they are removed out of the cell intact or were they degraded? So, and I mentioned the possibility that they could be shipped to a different organelle. And actually, in the case of Neman picthesis it's known that when you add cycloidextrin to the, that's the theory that Fred Maxli has. When you add cycloidextrin to the lysosome, somehow they fulfill the role of the missing Neman pic protein which was actually to move cholesterol from the lumen of the lysosome to the membrane of the lysosome so that it can be shipped out of the lysosome. So something like this may be happening also for A2E. Maybe they are sort of clustered in some kind of a crystal in the lumen of the, because these are hydrophobic compounds in the lumen of the lysosome and when you add the cycloidextrin you are helping it dissolve and maybe reach a different compartment in the cell, we don't know. So and also we've seen for example that we're interested in seeing where is the mechanism of cell death because that could give us an additional tool to use in the future. If we understand how the cells are being killed are they being killed by ER stress or by what is the process that is going on. We have additional ways to work downstream from cycloidextrin. Yes. Well I mean our friends, we became pretty good friends with Japanese chemists, they are producing our fluorescence cycloidextrin so we can really follow them as part of the project right now to see whether we can follow the distribution in the eye. Yeah, sure. Great. You mean stargars versus not in the eye or in the eye. How similar is the stargars labofucine versus the eye agents labofucine. Yeah, I don't know that there's a complete study on that but I think that many aspects of the stargars labofucine are similar in the sense they have A2E all trans the dimer all trans-radial dimer but there are other things also there's so-called melanolipofucine there are other components and I'm not sure really on how different they are and I'm not sure it's very well explained in the literature so yeah. Okay, thank you very much.